Induction of NADPH cytochrome P450 reductase by the Alzheimer beta-protein. Amyloid as a "foreign body".

A large body of data suggests that the Alzheimer's amyloid peptide (Abeta) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Abeta-mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Abeta peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non-physiological substrates (such as xenobiotics like drugs or other 'foreign molecules'), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Abeta can increase the expression of CP450r, SK-N-SH human neuroblastoma cells were exposed to Abeta25-35 and Abeta1-42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Abeta peptides, neuroblastoma cells showed a clear-cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non-transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged-matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Abeta causes oxidative stress.

Alzheimer beta protein mediated oxidative damage of mitochondrial DNA: prevention by melatonin.

Most contemporary progress in Alzheimer's disease (AD) stems from the study of a 42 43 amino acid peptide. called the amyloid beta protein (Abeta), as the main neuropathologic marker of the disorder. It has been demonstrated that Abeta has neurotoxic properties and that such effects are mediated by free-radicals. Exposure of neuronal cells to Abeta results in a spectrum of oxidative lesions that are profoundly harmful to neuronal homeostasis. We had previously shown that Abeta25-35 induces oxidative damage to mitochondrial DNA (mtDNA) and that this modality of injury is prevented by melatonin. Because Abeta25 35 does not occur in AD and because the mode of toxicity by Abeta25-35 may be different from that of Abeta1-42 (the physiologically relevant form of Abeta), we extended our initial observations to determine whether oxidative damage to mtDNA could also be induced by Abeta1-42 and whether this type of injury is prevented by melatonin. Exposure of human neuroblastoma cells to Abeta1-42 resulted in marked oxidative damage to mtDNA as determined by a quantitative polymerase chain reaction method. Addition of melatonin to cell cultures along with Abeta completely prevented the damage. This study supports previous findings with Abeta25-35, including a causative role for Abeta in the mitochondrial oxidative lesions present in AD brains. Most important, the data confirms the neuroprotective role of melatonin in Abeta-mediated oxidative injury. Because melatonin also inhibits amyloid aggregation, lacks toxicity, and efficiently crosses the blood-brain barrier, this hormone appears superior to other available antioxidants as a candidate for pharmacologic intervention in AD.

Inhibition of Alzheimer beta-fibrillogenesis by melatonin.

It is generally postulated that the amyloid beta protein (Abeta) plays a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathologic properties of this protein, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of Abeta to form beta-sheet structures or amyloid fibrils. We report that melatonin, a hormone recently found to protect neurons against Abeta toxicity, interacts with Abeta1-40 and Abeta1-42 and inhibits the progressive formation of beta-sheets and amyloid fibrils. These interactions between melatonin and the amyloid peptides were demonstrated by circular dichroism and electron microscopy for Abeta1-40 and Abeta1-42 and by nuclear magnetic resonance spectroscopy for Abeta1-40. Inhibition of beta-sheets and fibrils could not be accomplished in control experiments when a free radical scavenger or a melatonin analog were substituted for melatonin under otherwise identical conditions. In sharp contrast with conventional anti-oxidants and available anti-amyloidogenic compounds, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential new therapeutic agent in Alzheimer's disease.

Evidence of oxidative stress and in vivo neurotoxicity of beta-amyloid in a transgenic mouse model of Alzheimer's disease: a chronic oxidative paradigm for testing antioxidant therapies in vivo.

Increased expression of antioxidant enzymes and heat-shock proteins are key markers of oxidative stress. Such proteins are abnormally present within the neuropathological lesions of Alzheimer's disease (AD), suggesting that oxidative stress may play significant but yet undefined roles in this disorder. To gain further insight into the role of oxidative stress in AD, we studied the expression of CuZn superoxide dismutase (SOD) and hemoxygenase-1 (HO-1), two established markers of oxidative stress, in a transgenic mouse model of AD. Immunohistochemistry with anti-SOD and anti-HO-1 antibodies revealed a very pronounced increase of these proteins only in aged transgene-positive mice. Interestingly, the distribution of the oxidative burden was largely overlapping with dystrophic neuritic elements in the mice as highlighted with anti-ubiquitin antibodies. Because the most conspicuous alterations were identified around amyloid (Abeta) deposits, our results provide strong support for the hypothesis that Abeta is neurotoxic in vivo and that such toxicity is mediated by free radicals. To obtain additional experimental evidence for such an interpretation (ie, a cause-effect relationship between Abeta and oxidative neurotoxicity), PC12 cells were exposed to increasing concentrations of Abeta or to oxidative stress. In agreement with the in vivo findings, either treatment caused marked induction of SOD or HO-1 in a dose-dependent fashion. These results validate the transgenic approach for the study of oxidative stress in AD and for the evaluation of antioxidant therapies in vivo.

The amyloid beta protein induces oxidative damage of mitochondrial DNA.

Multiple lines of evidence suggest involvement of oxidative stress in the pathogenesis of Alzheimer disease (AD). The finding that amyloid beta peptide (A beta) has neurotoxic properties and that such effects are mediated in part by free-radicals has provided an avenue to explore new therapeutic strategies. In this study, we showed that exposure of PC 12 cells to an A beta fragment induces oxidative damage of mitochondrial DNA. Cells were exposed for 24 h to 50 microM A beta (25-35) or to 50 microM of a control peptide with a scrambled sequence. Oxidative damage of mitochondrial DNA (mtDNA) was assessed using a Southern blot technique and an mtDNA-specific probe recognizing a 13.5-kilobase restriction fragment. Treatment of DNA with NaOH was used to reveal abasic sites and single strand breaks. Treatment with endonuclease III or FAPy glycosylase was used to detect pyrimidine or purine lesions, respectively. Cells exposed to A beta exhibited marked oxidative damage of mtDNA as evidenced by characteristic changes on Southern blots. Cells exposed to the scrambled peptide did not show such modifications. Simultaneous addition of the pineal hormone melatonin consistently prevented the A beta-induced oxidative damage to mtDNA. Mitochondrial dysfunction in AD has been demonstrated by several laboratories. This study provides experimental evidence supporting a causative role of A beta in mitochondrial lesions of AD.

Anaplastic large cell lymphoma of T-cell phenotype in acquired immunodeficiency syndrome.

Anaplastic large cell lymphoma (ALCL) is an uncommon non-Hodgkin's lymphoma in the general population as well as in HIV-infected patients. Ordinarily, ALCL expresses T-cell phenotype, but lymphoproliferative disorders derived from T cells rarely occur in acquired immunodeficiency syndrome (AIDS). We describe a white male homosexual with AIDS who had bilateral pleural effusions. Examination of the pleural fluid revealed ALCL positive for Ki-1 (CD30), LCA (CD45), UCHL-1 (CD45RO), CD43, CD3, and epithelial membrane antigen. The lymphoma was negative for the B-cell marker L26 (CD20) and for Leu-M1 (CD15). The T-cell origin was also confirmed by the monoclonal rearrangement of the T-cell receptor beta chain gene. A review of other cases of ALCL in HIV-positive individuals shows variability in clinical presentation and biologic behavior of this lymphoma type. It also points to the potential contribution of gene rearrangement studies for recognition of phenotype. In addition, the role of determination of the presence of t(2;5) and the corresponding gene product is discussed.

Immunological detection and subcellular localization of Hsp70 and Hsp60 homologs in Trichomonas vaginalis.

Heat shock protein (Hsp) homologs of Trichomonas vaginalis belonging to Hsp70 and Hsp60 families with Mr 72 and 58 kDa, respectively, were detected by cross-reacting polyclonal antibody to DnaK of Escherichia coli. polyclonal antibody to Hsp60 of Heliothis virescens, and polyclonal antibody to GroEL/Hsp60 of cyanobacteria. A diffuse and finely granular intracellular staining of T. vaginalis was obtained with anti-DnaK on frozen 4-micron sections of the parasite as revealed by light microscopy. This antibody also recognized T. vaginalis antigen(s) present mainly in the Golgi apparatus, endoplasmic reticulum, and hydrogenosomes, as demonstrated by immunoelectron microscopy. Both antibodies against Hsp60 variants localized a homolog antigen in light microscopic granules, corresponding to hydrogenosomes of T. vaginalis.

The heat shock response and major heat shock proteins of Tritrichomonas mobilensis and Tritrichomonas augusta.

The responses to heat shock in Tritrichomonas mobilensis, a squirrel monkey parasite and Tritrichomonas augusta, an amphibian trichomonad, were evaluated by means of metabolic labeling with [35S]methionine. Electrophoretically separated trichomonad proteins synthesized at different temperatures were visualized by autoradiography and the label incorporation quantitated by a trichloroacetic acid precipitation procedure. A considerable difference in thermotolerance between the two species was found as the protein synthesis reached a maximum at 41 C in T. mobilensis and 37 C in T. augusta. The latter tolerated temperature increases 13 C above normal cultivation temperatures as compared to only 4 C thermotolerance range above normal in T. mobilensis. Major heat shock proteins (Hsps) were expressed in both T. mobilensis (with apparent Mr 94, 72, and 58 kDa) and T. augusta (Mr 94, 70, and 56 kDa) as revealed by autoradiography. Western blot analysis with polyclonal antibody against DnaK of Escherichia coli showed the presence of antigenic Hsp70 homologs in both trichomonads. Similarly, a polyclonal antibody against Hsp60 with broad interspecies cross-reactivity detected Hsp60 homologs in both T. mobilensis and T. augusta. The anti-DnaK antibody cross-reacted with a T. mobilensis protein localized in Golgi apparatus as demonstrated by immunoelectron microscopy. Immunocytochemistry on trichomonad frozen sections revealed the presence of the Hsp60 homolog in light-microscopic granules corresponding to hydrogenosomes.

Bilateral ulnar agenesis: case report and review of the literature.

Bilateral ulnar agenesis is a rare abnormality. A total of 36 cases are analyzed: 35 of these are documented in the literature and 1 stillborn male is presented in this study. Most patients had one of the three conditions: Al-Awadi/Raas-Rothschild syndrome, syndrome of ulnar aplasia with split hand/split foot deformity, or the Brachmann-de Lange syndrome. Fifty percent of all cases with bilateral ulnar agenesis were associated with lower limb defects and these cases, for the most part, also belonged to the aforementioned syndromes. Nonskeletal, internal organ malformations were identified in 34% of all patients. Nine patients presented with isolated bilateral ulnar agenesis. The Al-Awadi/Raas-Rothschild syndrome and the split hand/split foot deformity are heritable disorders. There was no evidence for genetic etiology in most of the other cases. Bilateral ulnar agenesis in our fetus was part of the Brachmann-de Lange syndrome with associated cardiac defect, diaphragmatic hernia, and umbilical artery agenesis.

Proteinases of Trichomonas vaginalis: antibody response in patients with urogenital trichomoniasis.

Immunoprecipitation combined with electrophoresis in gelatin-polyacrylamide gels was successfully used for detection of antibodies against numerous proteinases of Trichomonas vaginalis in infected patients. The method proved to be highly specific as anti-proteinase antibodies were absent in women with negative cultivation of T. vaginalis and no history of trichomoniasis. Sera of 71% and vaginal washes of 86% patients with trichomoniasis were positive for these antibodies. In vaginal washes, but not in sera, antibodies were partly complexed with proteinases, possibly of trichomonad origin. It was also shown that serum antibodies as well as local anti-proteinase antibodies persisted for weeks after patients had been cured.

Proteinases in Trichomonas vaginalis and Tritrichomonas mobilensis are not exclusively of cysteine type.

High molecular weight proteinases of Trichomonas vaginalis (with apparent Mr values 142 and greater than 220 kDa) and Tritrichomonas mobilensis (Mr 67, 86, 104 and 120 kDa), optimally active at pH8, were analysed in gelatin-containing polyacrylamide gels. All of these proteinases were resistant to serine-, aspartic- as well as cysteine proteinase inhibitors. Both proteolytic bands in T. vaginalis and two proteinases in T. mobilensis (67 and 104 kDa) were inhibited by EDTA and EGTA suggesting that they belong to the metallo-proteinase class. The 67 kDa proteinase of T. mobilensis was inhibited also by o-phenanthroline. The other two bands of T. mobilensis (86, 120 kDa) were not classified to any proteinase group since they appeared to be resistant to the chelating agents tested in this study.

Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax.

Proteolytic activities in crude extracts and culture filtrates from Trichomonas tenax were determined using hide powder azure as substrate and the proteinase profiles in both samples were analysed in SDS-polyacrylamide gels containing copolymerized gelatin. The enzyme activity in the crude extract was detected over a broad pH range and was strongly activated by dithiothreitol, mainly in the pH range 5-8, and inhibited by cysteine proteinase inhibitors. Extracellular enzyme activity in culture filtrates was SH-dependent and increased continuously during incubation of the cell suspension, suggesting proteinase release. A total of seven distinct proteolytic bands could be detected in crude preparations. Three of these, with apparent Mr values 35,000, 45,000 and 56,000 and a pH optimum of 4-7, were SH-dependent and their inhibitory sensitivities were characteristic for cysteine proteinases. The 45,000 and 56,000 proteinases probably corresponded to those found in the culture filtrates. Proteolytic bands with apparent Mr 76,000, 87,000, 102,000 and 270,000 and pH optima in the alkaline region, pH 8-9, were independent of SH groups and were inhibited by a chelating agent EDTA, suggesting that they belong to the metalloproteinase family.

Degradation of collagen types I, III, IV and V by extracellular proteinases of an oral flagellate Trichomonas tenax.

Proteinases secreted by an axenic strain of Trichomonas tenax were active against native types I, III, IV and V collagens when evaluated by polyacrylamide gel electrophoresis. Degradation of all four collagen types was temperature dependent. Basement membrane type IV collagen was digested most effectively. An inhibition of all collagenolytic activities by a specific inhibitor of cysteine proteinases, E-64, and activation by a reducing agent, dithiothreitol, indicated the involvement of cysteine proteinases of the oral flagellate in the cleavage of collagen.

Proteolytic activity in Tritrichomonas mobilensis.

Cell extracts of an entero-invasive protozoon of squirrel monkeys, Tritrichomonas mobilensis, contained relatively high proteolytic activity, measured on hide powder azure (HPA). Multiple proteinase forms, optimally active at pH 5-7, were detected by electrophoretic analysis in gelatin-containing polyacrylamide gels. Three major proteinase bands of apparent low molecular weights, Mr 18, 23 and 30 kDa, were seen on gels. Inhibition-activation studies suggest that only cysteine proteinases were involved in HPAase and gelatinolytic activities of T. mobilensis cell extracts.