Environmental metabolomics uncovers oxidative stress, amino acid dysregulation, and energy impairment in Daphnia magna with exposure to industrial effluents.

Anthropogenic activities are regarded as point sources of pollution entering freshwater bodies worldwide. With over 350,000 chemicals used in manufacturing, wastewater treatment and industrial effluents are comprised of complex mixtures of organic and inorganic pollutants of known and unknown origins. Consequently, their combined toxicity and mode of action are not well understood in aquatic organisms such as Daphnia magna. In this study, effluent samples from wastewater treatment and industrial sectors were used to examine molecular-level perturbations to the polar metabolic profile of D. magna. To determine if the industrial sector and/or the effluent chemistries played a role in the observed biochemical responses, Daphnia were acutely (48 h) exposed to undiluted (100%) and diluted (10, 25, and 50%) effluent samples. Endogenous metabolites were extracted from single daphnids and analyzed using targeted mass spectrometry-based metabolomics. The metabolic profile of Daphnia exposed to effluent samples resulted in significant separation compared to the unexposed controls. Linear regression analysis determined that no single pollutant detected in the effluents was significantly correlated with the responses of metabolites. Significant perturbations were uncovered across many classes of metabolites (amino acids, nucleosides, nucleotides, polyamines, and their derivatives) which serve as intermediates in keystone biochemical processes. The combined metabolic responses are consistent with oxidative stress, disruptions to energy metabolism, and protein dysregulation which were identified through biochemical pathway analysis. These results provide insight into the molecular processes driving stress responses in D. magna. Overall, we determined that the metabolic profile of Daphnia could not be predicted by the chemical composition of environmentally relevant mixtures. The findings of this study demonstrate the advantage of metabolomics in conjunction with chemical analyses to assess the interactions of industrial effluents. This work further demonstrates the ability of environmental metabolomics to characterize molecular-level perturbations in aquatic organisms exposed to complex chemical mixtures directly.

Swelling of nuclei and decondensation of chromatin in a multinuclear model.

Rat liver nuclei were isolated in the presence of a nonionic detergent, and the soluble proteins were extracted from them using low-salt solution. The isolated nuclei were then embedded in a fibrin clot which was cut into 4 to 5 mm3 pieces and placed into an AMICON pressure cell in the presence and absence of the soluble nuclear proteins. When the soluble nuclear proteins were present and when their concentrations reached 40 mg/ml, the nuclei were significantly larger and their chromatin more decondensed than in the absence of added proteins. Our findings suggest that the soluble nuclear proteins play a role in the regulation of nuclear volume and the hydration of chromatin.

The effects of additional pathology on the cognitive deficit in Alzheimer disease.

The diagnosis of Alzheimer disease (AD) according to current criteria is a combined clinical and pathological exercise. The clinical discrimination of AD from other types of dementia may be complicated when the patient suffers from more than one disease. In particular the concomitant presence of other neurological conditions may significantly influence the severity of cognitive deficit. In this study we analyze the extent of the influence of vascular and other neurodegenerative pathology on the cognitive deficit in a consecutive series of 88 prospectively assessed elderly subjects. We find that, for any given level of cognitive deficit, the densities of either all plaques or neuritic plaques alone in the neocortex are significantly lower in cases of AD mixed with other CNS pathology than in cases of AD with no other CNS pathology. In AD combined with cerebrovascular disease, the total plaque density makes a significant contribution to cognitive deficit, while neurofibrillary tangle (NFT) densities do not. In contrast, in pure AD tangle density is the major determinant of cognitive deficit. Our findings draw attention to the influence of coexisting brain pathologies on the clinical manifestation of dementia in subjects with AD. These findings indicate that pathological diagnostic criteria for AD should take into account such additional pathology in demented subjects. They also improve understanding of the circumstances in which the amyloid component of AD can play a decisive role in precipitating clinical dementia.

Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease.

BACKGROUND: Recent studies suggest that vascular disease may contribute to the cause of Alzheimer disease (AD). Since elevated plasma total homocysteine (tHcy) level is a risk factor for vascular disease, it may also be relevant to AD. OBJECTIVE: To examine the association of AD with blood levels of tHcy, and its biological determinants folate and vitamin B12. DESIGN: Case-control study of 164 patients, aged 55 years or older, with a clinical diagnosis of dementia of Alzheimer type (DAT), including 76 patients with histologically confirmed AD and 108 control subjects. SETTING: Referral population to a hospital clinic between July 1988 and April 1996. MAIN OUTCOME MEASURES: Serum tHcy, folate, and vitamin B12 levels in patients and controls at entry; the odds ratio of DAT or confirmed AD with elevated tHcy or low vitamin levels; and the rate of disease progression in relation to tHcy levels at entry. RESULTS: Serum tHcy levels were significantly higher and serum folate and vitamin B12 levels were lower in patients with DAT and patients with histologically confirmed AD than in controls. The odds ratio of confirmed AD associated with a tHcy level in the top third (> or = 14 micromol/L) compared with the bottom third (< or = 11 micromol/L) of the control distribution was 4.5 (95% confidence interval, 2.2-9.2), after adjustment for age, sex, social class, cigarette smoking, and apolipoprotein E epsilon4. The corresponding odds ratio for the lower third compared with the upper third of serum folate distribution was 3.3 (95% confidence interval, 1.8-6.3) and of vitamin B12 distribution was 4.3 (95% confidence interval, 2.1-8.8). The mean tHcy levels were unaltered by duration of symptoms before enrollment and were stable for several years afterward. In a 3-year follow-up of patients with DAT, radiological evidence of disease progression was greater among those with higher tHcy levels at entry. CONCLUSIONS: Low blood levels of folate and vitamin B12, and elevated tHcy levels were associated with AD. The stability of tHcy levels over time and lack of relationship with duration of symptoms argue against these findings being a consequence of disease and warrant further studies to assess the clinical relevance of these associations for AD.

SPECT perfusion imaging in the diagnosis of Alzheimer's disease: a clinical-pathologic study.

OBJECTIVE: Numerous studies have suggested that temporoparietal hypoperfusion seen on brain imaging with SPECT may be useful in diagnosing AD during life. However, these studies have often been limited by lack of pathologic validation and unrepresentative samples. The authors performed this study to determine whether SPECT imaging provides diagnostically useful information in addition to that obtained from a clinical examination. METHODS: Clinical data and SPECT images were collected prospectively, and patients were followed to autopsy. Clinical history, pathologic findings, and SPECT images were each evaluated by raters blind to other features, and clinical and SPECT diagnoses were compared with pathologic diagnoses. The study population consisted of 70 patients with dementia, followed to autopsy; 14 controls followed to autopsy; and 71 controls (no autopsy performed). The primary outcome was the likelihood of a pathologic diagnosis of AD given a positive clinical diagnosis, a positive SPECT diagnosis, and both. RESULTS: When all participants (patients and controls) were included in the analysis, the clinical diagnosis of "probable" AD was associated with an 84% likelihood of pathologic AD. A positive SPECT scan raised the likelihood of AD to 92%, whereas a negative SPECT scan lowered the likelihood to 70%. SPECT was more useful when the clinical diagnosis was "possible" AD, with the likelihood of 67% without SPECT, 84% with a positive SPECT, and 52% with a negative SPECT. Similar results were found when only patients with dementia were included in the analysis. CONCLUSIONS: In the evaluation of dementia, SPECT imaging can provide clinically useful information indicating the presence of AD in addition to the information that is obtained from clinical evaluation.

Influence of the apolipoprotein E genotype on amyloid deposition and neurofibrillary tangle formation in Alzheimer's disease.

The effect of the apolipoprotein E genotype on the development of late onset Alzheimer's disease is still debated. Neuropathological studies of Alzheimer's disease have found a great extent of amyloid deposition in cortex and blood vessel walls in association with the apolipoprotein E epsilon 4 genotype [Rebeck G. W. et al. (1993) Neuron 11, 575-580; Schmechel et al. (1993) Proc. natn. Acad. Sci. U.S.A. 90, 9649-9653]. In contrast, the relationship of apolipoprotein E genotype to neurofibrillary pathology in Alzheimer's disease has been less clear. In this study we present evidence on the influence of the apolipoprotein E genotype on Alzheimer's disease related pathology in a series of 76 autopsy cases that had pathology that fulfilled the CERAD criteria for Alzheimer's disease. We found that the presence of the apolipoprotein E epsilon 4 allele is correlated with increased amounts of both amyloid and neuritic pathology in the neocortex as determined using an image analysis system. Comparison of plaque and tangle densities with the allele doses of epsilon 2 and epsilon 4 revealed a striking parallelism, suggesting that the alleles exert their effects very early in the pathological process before deposition of plaques and tangles. Although the apolipoprotein E epsilon 2 allele had a protective effect against both amyloid deposition and neurofibrillary tangle formation, in the presence of the epsilon 4 allele this protective effect against neuritic pathology was less marked than against amyloid deposition. This differential effect on amyloid deposition and the accumulation of neuritic pathology suggests that different molecular mechanisms are involved in the effect of apolipoprotein E on amyloid deposition and on tau phosphorylation.

The diagnosis of Alzheimer's disease: a question of image?

The most common cause of dementia in the developed world is Alzheimer's disease. Histopathology is required to confirm diagnosis, but most evaluations of the accuracy of clinical criteria and neuroimaging in the diagnosis of dementia of the Alzheimer type are without such confirmation. The average specificity of clinical criteria alone is about 75%. This paper discusses the contribution of simple structural (x-ray computed tomography [CT]) and functional (Tc-99m-HMPAO single photon emission computed tomography [SPECT]) imaging to the diagnosis of Alzheimer's disease in 71 histopathologically confirmed cases (47 with Alzheimer's disease, 16 with other dementias, 8 controls) and 84 living controls. Medial temporal lobe atrophy assessed by temporal lobe-oriented CT gave 94% sensitivity and 93% specificity, while parietotemporal hypoperfusion on SPECT revealed 96% sensitivity and 89% specificity. The combination of both changes yielded a sensitivity of 90% and a specificity of 97%. These investigations clearly enhance diagnostic accuracy, can be readily applied in the clinical situation, and could be used in epidemiologic studies of Alzheimer's disease.

Mannan-binding lectin in human serum, cerebrospinal fluid and brain tissue and its role in Alzheimer's disease.

Mannan-Binding lectin (MBL) is a serum lectin which can activate the classical complement pathway. Complement proteins of the classical pathway have been found in the brains of patients with Alzheimer's disease (AD) in association with AD brain pathology. To investigate the role for MBL in AD we have looked for its presence in the brain by immunohistochemistry and determined the levels of MBL in paired samples of cerebrospinal fluid and serum from AD patients and controls. MBL was detected in association with blood vessels in the brain tissue of both AD patients and control subjects. There was no apparent difference in the distribution of MBL in the brain tissue between the two groups. The mean concentration of MBL in the CSF was 44% lower in AD patients than in controls (AD 154 +/- 35 pg/ml, n = 19; non-AD 276 +/- 50 pg/ml, n = 15, p < 0.05). The levels of MBL in serum were not significantly different in the two groups. Thus, this study shows that MBL is associated with blood vessels in the brains of both AD and control subjects. Moreover, CSF levels of MBL appear to be lower in AD patients than in control subjects which may indicate a higher degree of MBL consumption connected with complement activation in the AD patients.

Differences in nonenzymatic glycation of histones.

Time course of glucose binding by histone Hl and total histones was followed in isolated histone preparations and in thymus nuclei. In both cases the uptake of glucose by Hl was surprisingly high in contrast to a much lower uptake of glucose by total histones. DNA is not implicated in glycation of histones in nuclei.

Low-molecular-weight zinc-binding proteins in liver cytosol of the allylisopropylacetamide (AIA) treated rats.

Rats maintained on a normal diet supplemented with 10 microCi 65Zn/day for a period of 10 days were given allylisopropylacetamide (AIA), 400 mg/kg body weight: 24 h later they were killed. Cytosol was prepared from liver cells and analysed by gel filtration on a Sephadex G-75 column. The AIA treatment caused an alteration in the chromatographic pattern of Zn-binding proteins, and a low-molecular-weight fraction appeared.

Myelinated axon number in the optic nerve is unaffected by Alzheimer's disease.

AIMS/BACKGROUND: Visual symptoms are a common but not invariable feature of Alzheimer's disease (AD) and such symptoms appear to become more pronounced as the severity of the dementia increases. Pathology in both the pregeniculate and cortical parts of the visual system has been suggested to underlie the visual deficits in AD. In order to investigate the former possibility, the effect of AD on the optic nerve was investigated. METHODS: Intraorbital segments of optic nerve were taken at autopsy from nine patients with AD and seven patients with no history of psychiatric or neurological disease and no abnormal neuropathology. All patients had functional vision before death and appeared free of retinal, optic nerve, or microvascular disease. The optic nerves were processed into resin, semi-thin sections cut perpendicular to the long axis of each optic nerve, and stained with paraphenylenediamine. The sections were then investigated using an image analysis system and standard morphometric techniques. RESULTS: There was no significant difference in the mean cross sectional neural area of AD compared with control optic nerves. Neither were there any significant differences between myelinated axon surface density, total axon number, or mean cross sectional axon area in AD compared with control optic nerves. CONCLUSION: These results indicate that optic nerve degeneration is not a feature of AD and suggest that the visual deficits in the disease result from cortical dysfunction. This view is supported by the fact that visuospatial dysfunction appears to be the most common visual problem in AD.

Acupuncture for chronic asthma.

BACKGROUND: Acupuncture has traditionally been used to treat asthma in China and is used increasingly for this purpose internationally. OBJECTIVES: The objective of this review was to assess the effects of acupuncture for the treatment of asthma or asthma-like symptoms. SEARCH STRATEGY: We searched the Cochrane Airways Group trials register, the Cochrane Complementary Medicine Field trials register and reference lists of articles. SELECTION CRITERIA: Randomised and possibly randomised trials using acupuncture to treat asthma and asthma-like symptoms. Acupuncture could involve the insertion of needles or other forms of stimulation of acupuncture points. DATA COLLECTION AND ANALYSIS: Trial quality was assessed by at least two reviewers independently. A reviewer experienced in acupuncture assessed the adequacy of the sham acupuncture. Study authors were contacted for missing information. MAIN RESULTS: Seven trials involving 174 people were included. Trial quality varied and results were inconsistent. No statistically significant or clinically relevant effects were found for acupuncture compared to sham acupuncture. However the points used in the sham arm of some studies are used for the treatment of asthma according to traditional Chinese medicine. Only one study used individualised treatment strategies. Lung function could be compared statistically in only 3 trials. Peak expiratory flow rate showed a statistically insignificant increase of 8.4 litres/minute weighted mean difference (95% confidence interval -29.4 to 46.2) when acupuncture was compared to sham acupuncture. REVIEWER'S CONCLUSIONS: There is not enough evidence to make recommendations about the value of acupuncture in asthma treatment. Further research needs to consider the complexities and different types of acupuncture.

Homeopathy for chronic asthma.

BACKGROUND: Homeopathy involves the use, in dilution, of substances which cause symptoms in their undiluted form. It is one of the most widespread forms of complementary medicines and is also used to treat asthma. OBJECTIVES: The objective of this review was to assess the effects of homeopathy in people with chronic stable asthma. SEARCH STRATEGY: We searched the Cochrane Airways Group trials register, the Cochrane Complementary Medicine Field trials register, the Glasgow Homeopathic Hospital database, the Muenchener Modell database and reference lists of articles. SELECTION CRITERIA: Randomised and possibly randomised trials of homeopathy for the treatment of stable chronic asthma, with observation periods of at least one week. DATA COLLECTION AND ANALYSIS: Data extraction was done by one reviewer and checked by the second reviewer. Trial quality was assessed by the reviewers. MAIN RESULTS: Three trials with a total of 154 people were included. These trials were all placebo-controlled and double-blind, but of variable quality. They used three different homeopathic treatments which precluded quantitative pooling of results. The standardised treatments in these trials are unlikely to represent common homeopathic practice, where treatment tends to be individualised. In one trial, severity of symptoms was lessened in the homeopathy group compared to the placebo group. In another trial, lung function measures and medication use showed improvement in the homeopathy group compared to the placebo group. The third trial found improvement in both the homeopathy and placebo groups, but no difference between the groups. REVIEWER'S CONCLUSIONS: There is not enough evidence to reliably assess the possible role of homeopathy in asthma. As well as randomised trials, there is a need for observational data to document the different methods of homeopathic prescribing and how patients respond.

Identification of nucleohistones by glycosylation and basic dyes.

Earlier examinations have shown that reducing sugars mainly react with the epsilon-amino group of lysine. After DNA extraction, lysine-rich histone proteins of alcohol fixed nuclei of rat organs were treated with glucose-6-phosphate. It was possible to selectively stain the histone type proteins of DNA negative nuclei with basic dyes.

Standards for reporting interventions in controlled trials of acupuncture: the STRICTA recommendations.

Acupuncture treatment and control group interventions in parallel-group randomised trials of acupuncture are not always precisely reported. In an attempt to improve standards, an international group of experienced acupuncturists and researchers devised a set of recommendations, designating them STRICTA: STandards for Reporting Interventions in Controlled Trials of Acupuncture. In a further consensus-building round, the editors of several journals helped redraft the recommendations. These follow the Consolidated Standards for Reporting Trials (CONSORT) format, acting as an extension of the CONSORT guidelines for the specific requirements of acupuncture studies. Participating journals are publishing the STRICTA recommendations and requesting prospective authors to adhere to them when preparing reports for publication. Other journals are invited to adopt these recommendations. The intended outcome is that interventions in controlled trials of acupuncture will be more adequately reported, thereby facilitating an improvement in critical appraisal, analysis and replication of trials.

The reaction of reducing sugars with histones.

The epsilon amino groups of histone proteins were eliminated by condensation with glucose, fructose or mannose. The trichloroacetic acid extracted, DNA negative nuclei treated with reducing sugars, stained easily with basic dyes.

Identification of basic nuclear proteins by their boronate complex.

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.

A critical analysis of acupuncture in pulmonary disease: efficacy and safety of the acupuncture needle.

Criteria for therapeutic efficacy and safety include significant amelioration of symptoms and, ideally, cure (i.e., patients' belief in effective improvement of symptoms and quality of life, durable impact on symptoms, verifiable subjective and objective changes); improved patient management (e.g., diminishing, or ceasing medication, physiotherapy, and other interventions); safety for patient and practitioner and an acceptable side effect profile; cost-effectiveness of the therapy in practice and to teach to others. There is evidence that in bronchial asthma, chronic bronchitis, and chronic disabling breathlessness the use of acupuncture fulfills these to varying degrees. It can facilitate reducing pharmacologic medication and is safe, suggesting that acupuncture as an adjuvant in the treatment of respiratory disease might be safer than prolonged pharmaceutical maintenance therapy alone. Its cost-effectiveness has yet to be adequately researched. Twenty-one papers in English were obtained and 16 were further evaluated; eight were double-blind, five single-blind, and three unblinded. The remaining five, and most of the Chinese literature, were excluded on account of their poor quality. Acupuncture was effective in four of eight of the double-blind, three of five single-blind, and three of three unblinded studies (i.e., 10 of 16 [62.5%] overall). A previously unreported confounding variable was identified and concerned the designation of sham points. Most sham points were believed to be inactive but, according to traditional Chinese principles, many are active in pulmonary disease. Reappraised accordingly, the unequivocally positive studies were summed with those in which "real" and "sham" acupuncture were not significantly different but in which the combined effect of all acupuncture (i.e., real + sham) on breathlessness was significantly different from baseline. This yielded 13 of 16 (81%) [corrected] studies in which acupuncture led to significant improvement. In most studies, current pharmacologic treatment had a greater effect than acupuncture alone. However, in the 11 studies in which it was evaluated, medication could be significantly reduced by acupuncture in 10 (91%). Twenty-three of the 320 patients in the 16 studies (7%) reported minimal side effects, none requiring intervention. Current published evidence reveals no reason to withhold acupuncture as a safe and potentially effective treatment in patients with bronchial asthma and chronic obstructive lung disease. Further, more appropriately designed studies are urgently required. This would be facilitated in the United States by licensing the acupuncture needle as a therapeutic agent and might lead to important new insights and therapeutic opportunities.

Diseases of meaning, manifestations of health, and metaphor.

Disease and health are commonly thought of as distinct opposites. We propose a different view in which both may be seen to be facets of healthy functioning, each necessary for the other, each giving rise to the other. Thus, disease may be thought of as a manifestation of health. It is the healthy response of an organism striving to maintain physical, psychologic, and spiritual equilibrium. Disease is not necessarily to be avoided, blocked, or suppressed. Rather, it should be understood to be a process of transformation. The process should therefore be facilitated because it is an integral part of the dynamic equilibrium that we ordinarily think of as health. In many cases, perhaps all, people get ill because there is something going "wrong" in their lives. This could occur in a whole range of ways-relationships, environment, food, or job. Our view, however, is that disease is a meaningful state that can inform health workers how to help patients to heal themselves. In this way, instead of being meaningless, people's problems become diseases of meaning, enabling people to see that things are not necessarily "going wrong" but are, in fact, helping them become stronger, to live more fully and with more understanding. Seen from this perspective, depression; cancer; heart disease; neurodegenerative and autoimmune disease; dementia; and conditions such as community violence, genocide, and the problem of environmental devastation are "diseases of meaning." World Health Organization forecasts make it clear that diseases of meaning will continue well into the next millennium to be the major cause of suffering and death worldwide. To deal with them, the world needs to reformulate the biomolecular paradigm that has been exploited in the last two centuries. It does not address the reasons why these diseases arise, attending mainly to their molecular consequences. A paradigm that includes the importance of meaning must now be given top priority. The concept that diseases are a manifestation of health-a call to a different relationship with ourselves and our environment, both animate and inanimate- is in itself a different approach. Programs for care and education based upon it would have immediate application in medicine, industry, education and ecology. We believe that this model would have far-reaching consequences for the understanding, treatment, and prevention of diseases and behaviors that lead to violence and environmental destruction.

The bulk of ATP is associated to proteins in the living cell: a release kinetics study.

The analysis of the nonionic detergent induced release kinetics of intracellular components is a promising approach to obtain information on the possible molecular interactions inside the living cell. The molecular interactions of ATP were studied by monitoring the nonionic detergent-induced release kinetics of ATP from cultured human cells. The observed rapid mobilization rate in the presence of Triton X 100 and the retarded release in the presence of Brij 58 were in good agreement with the detergent-induced release kinetics of proteins and K+ reported earlier. These data are consistent with the idea that the bulk of ATP is associated to the protein macromolecules of the living cell.