The budding yeast, Saccharomyces cerevisiae, as a model for aging research: a critical review

In this review we discuss the yeast as a paradigm for the study of aging. The budding yeast Saccharomyces cerevisiae, which can proliferate in both haploid and diploid states, has been used extensively in aging research. The budding yeast divides asymmetrically to form a ‘mother’ cell and a bud. Two major approaches, ‘budding life span’ and ‘stationary phase’ have been used to determine ‘senescence’ and ‘life span’ in yeast. Discrepancies observed in metabolic behavior and longevity between cells studied by these two systems raise questions of how ‘life span’ in yeast is defined and measured. Added to this variability in experimental approach and results is the variety of yeast strains with different genetic make up used as ‘wild type’ and experimental organisms. Another problematic genetic point in the published studies on yeast is the use of both diploid and haploid strains. We discuss the inherent, advantageous attributes that make the yeast an attractive choice for modern biological research as well as certain pitfalls in the choice of this model for the study of aging. The significance of the purported roles of the Sir2 gene, histone deacetylases, gene silencing, rDNA circles and stress genes in determination of yeast ‘life span’ and aging is evaluated. The relationship between cultivation conditions and longevity are assessed. Discrepancies between the yeast and mammalian systems with regard to aging are pointed out. We discuss unresolved problems concerning the suitability of the budding yeast for the study of basic aging phenomena.     

Conceptual issues involved with the genetics of Alzheimer''s disease

Although Davies succinctly discusses the technical issues surrounding a possible genetic role in Alzheimer's disease, important conceptual issues require careful consideration. One involves the general assumption that plaques and tangles represent the critical qualitative variable for defining patients with Alzheimer's disease, and the effect that deviations from this assumption will have on genetic studies. Another issue involves the value of research strategies based on ‘single gene’ or ‘gene candidate’ concepts, given the probable multifactorial characteristic of Alzheimer's disease.     

Optimization of a protein synthesizing lysate system from Trypanosoma cruzi

A protein-synthesizing lysate system from Trypanosoma cruzi epimastigotes analogous to the rabbit reticulocyte lysate system was established. The system was optimized by the ‘classical’ method where one of the factors is varied while the others are kept constant. With this the following optima were found: [Mg²⁺]: 1.0 mM, [K⁺]: 60 mM, T: 25°C, pH: 7.5. This method was compared with the ‘sequential simplex’ method [Long, D.E. (1969) Anal. Chim. Acta 46, 93–100], a method designed to optimize rationally interdependent factors in biological systems. The optima as determined with this method were: [Mg²⁺]: 1.02 mM, [K⁺]: 63 mM, T: 25.5°C, pH: 7.25. At these values the system incorporated 43% more amino acids into proteins than a system optimized with the ‘classical’ method. Fluorographic analysis of the proteins synthesized by the system shows that while proteins in the molecular weight range between 14 000 and 45 000 are synthesized in amounts comparable to the in vivo situation, the higher molecular weight proteins (>45 000) are synthesized in lesser quantities.     

The diagnosis related groups enhanced electronic medical record

Problem. The introduction of Diagnosis Related Groups as a basis for hospital payment in Germany announced essential changes in the hospital reimbursement practice. A hospital's economical survival will depend vitally on the accuracy and completeness of the documentation of DRG relevant data like diagnosis and procedure codes. In order to enhance physicians’ coding compliance, an easy-to-use interface integrating coding tasks seamlessly into clinical routine had to be developed. A generic approach should access coding and clinical guidelines from different information sources. Methods. Within the Electronic Medical Record (EMR) a user interface (‘DRG Control Center’) for all DRG relevant clinical and administrative data has been built. A comprehensive DRG-related web site gives online access to DRG grouping software and an electronic coding expert. Both components are linked together using an application supporting bi-directional communication. Other web based services like a guideline search engine can be integrated as well. Results. With the proposed method, the clinician gains quick access to context sensitive clinical guidelines for appropriate treatment of his/her patient and administrative guidelines for the adequate coding of the diagnoses and procedures. This paper describes the design and current implementation and discusses our experiences.     

Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients

Several aspects of enzyme-linked immunosorbent assay (ELISA) procedures and data analysis have been examined in an attempt to find a rapid and reliable method for discriminating between ‘positive’ and ‘negative’ results when testing a large number of samples. A layout of ELISA plates was designed to reduce uncontrolled variation and to optimize the number of negative and positive controls. A transformation using the fourth root (A^1/4) of the optical density readings corrected for the blank (A) stabilized the variance of most ELISA data examined. Transformed A values were used to calculate the true limits, at a set protection level, for false positive (C) and false negative (D). Methods are discussed to reduce the number of undifferentiated samples, i.e. the samples with response falling between C and D. The whole procedure was set up for use with an electronic spreadsheet. With the addition of few instructions of the type ‘if…then…else’ in the spreadsheet, the ELISA results were obtained in the simple trichotomous form ‘negative/undefined/positive’. This allowed rapid analysis of more than 1100 maize samples testing for the presence of seven aphid-borne viruses—in fact almost 8000 ELISA samples.     

Thyroid disorders: Automatic diagnosis in CONSULT I

Automatic diagnosis of thyroid diseases is implemented on CONSULT I^™, a microcomputer system based on the Patrick model for computer-assisted diagnosis in medicine. The thyroid ‘subsystem’ consists of 19 classes (diseases) and 16 features (signs, symptoms, laboratory tests). For 76 test cases obtained from patient records (recognition samples), the ‘true’ class (disease) is decided in the highest ‘probability’ number in 89% of cases and in the differential diagnosis in 100% of cases. Performance is compared to physicians. Estimation of class-conditional probability densities utilizing equivalence regions in the feauture space is discussed.     

Progress toward a new injury severity characterization: Severity profiles

Presented is a new seven-dimensional injury severity profile. The profile includes three physiologic assessments and four variables which express the number, location, and severity of a patient's injuries in terms of ‘Abbreviated injury scale’ values. The physiologic assessments are coded values for the ‘Glasgow coma scale’, systolic blood pressure, and respiratory rate.

Also presented are survival-death predictive values of a cluster model based on survival rates of clusters of profiles of 2569 blunt-injured and penetrating-injured patients. The cluster model has a relative information gain (R) of 0.90. R is a measure of predictive value relative to an infallible predictor. It varies from 0 to 1, the higher the value the better the predictive value. The model had 26 false negatives (deaths predicted to survive) and 35 false positives (survivors predicted to die) giving rise to a false negative rate of 9.3%, a false positive rate of 1.4% and a misclassification rate of 2.4%. The R value and false negative rate are particularly noteworthy, the R value being higher than, and the false negative rate much lower than typical values of 30–40% achieved by TRISS (a combination index based on trauma score, injury severity score and patient age).

Also noteworthy is that the clustering was independent of survival/death outcome information and that the good results were achieved even though patient age has not yet been incorporated into the model.     

Capacity to consent to research among patients with bipolar disorder

Experts have debated the influence of mental illness on decision-making capacity. This paper reviews concepts of decision-making capacity and existing research on the influence of mental illness on capacity to consent to research. We propose how bipolar disorder, especially mania, may have an effect on consent capacity. The current conceptualization of capacity utilizes legal standards of ‘choice’, ‘understanding’, ‘appreciation’ and ‘rational reasoning’, as well as voluntarism, or the assurance that the patient is free to agree or to decline to participate in research. Studies of patients with schizophrenia suggest impaired cognition influences ‘understanding’ and is more important than severity of psychosis in affecting decision-making abilities. There are no studies of sources and extent of impairment to consent to research among manic patients. Mania may influence a patient’s understanding of the research protocol, but also alter the patient’s views, values and level of insight, thus impairing decision-making abilities at the ‘appreciation’ standard even when the patient understands the relevant information. Mania may impact freedom to decide, yet paradoxically, manic patients may be less influenced by others and less vulnerable to coercion, undue influence and undue incentives compared to patients without mental illness. We suggest that in patients with mood disorders, the legal standard of appreciation be thoroughly probed during the consent procedure. Studies of the effect of mania and depression on consent capacity and voluntarism are needed in order to develop processes that increase safeguards in the informed consent process.     

Long-term potentiation in the hippocampus

Long-term potentiation of field and single neuronal responses recorded in various hippocampal fields is described on the basis of author's and literary data. Most of intrahippocampal and extrinsic connections in both in vivo and in vitro hippocampal preparations show this phenomenon after one or several conditioning trains of comparatively short duration (20 s or less) at various frequencies (from 10 to 400 Hz). Properties of hippocampal potentiation are described. The properties include long term persistence (hours and days) of the potentiated response, its low frequency depression, self-restoration after the depression, specificity of the potentiation for the tetanized pathway, necessity of activation of a sufficient number of neuronal elements (‘cooperativity’) to produce the potentiation, possible involvement of ‘reinforcing’ brain structures during conditioning tetanization. These properties are distinct from those of ‘usual’ short-term post-tetanic potentiation and lead to the suggestion that the neuronal mechanisms underlying long-term potentiation are similar to those underlying memory and behavioralconditioned reflex. Neurophysiological mechanisms of long-term potentiation are discussed. The main mechanism consists in an increase in efficacy of excitatory synapses as shown by various methods including intracellular recording and quantal analysis. The latter favours presynaptic localization of the changes of synaptic efficacy showing increase in the number of transmitter quanta released per presynaptic impulse. However, changes in the number of subsynaptic receptors or localized changes in dendritic postsynaptic membrane are not excluded. Biochemical studies indicate the increase in transmitter release and calcium-dependent phosphorylation of pyruvate dehydrogenase after tetanization. Instances of persistent response facilitations at other levels of the vertebrate central nervous system (especially at neocortical level) are considered and compared with hippocampal long-term potentiation.

It is suggested that modifiable excitatory synapses necessary for learning have been identified in studies of long-term potentiation. These synapses are presumably modified as a result of close sequential activation of the following three structures: excitatory presynaptic fibers, the postsynaptic neuron and a ‘reinforcing’ brain system.     

Confirmatory tests for human T-cell leukemia virus type 1 (HTLV-1): Western blot compared with RIPA on African sera

Confirmation of human T-Cell leukemia virus type 1 (HTLV-1) seropositivity calls for reactivity against at least 2 proteins encoded by 2 different genes, revealed by Western blot (WB) and /or radioimmuno-precipitation assay (RIPA). To evaluate the use of WB as a basis for applying these criteria, we conducted a study of two types of WB and compared them with RIPA patterns. The first part of the work, performed with 40 African sera, used Dupont de Nemours commercialized WB and an ‘in-house’ WB. Both WB detected antibody to proteins encoded by 2 different genes: antibody to gag products were revealed equally by both WB, but commercialized WB detected antibody to tax protein whereas the ‘in-house’ WB detected antibody to env protein (gp46) more efficiently.

The second part of the work, conducted with 158 African sera, compared results of an ‘in-house’ virus lysate WB and RIPA. Our data show a perfect concordance between the two procedures when sera were clearly positive by WB (gag + env reaction). Sera reacting to p19 and p24 (both gag) by WB were confirmed positive by RIPA in 75% of the cases. The majority of the indeterminate WB profiles not confirmed by RIPA presented isolated gag reactivity (p15 or p19 or p24).     

Open systems: panoramic views of gene expression

Since their development in the early 1990s, differential gene expression (DGE) technologies have been applied to a multitude of biological challenges, both for the purpose of basic biological research and as a valuable tool for the discovery and development of pharmaceuticals. In this review we survey a class of DGE technologies collectively referred to as ‘open’ architecture systems. These technologies are distinct from the ‘closed’ DGE technologies (quantitative PCR, chip technologies), in that no pre-existing biological or sequence information is necessary and they are applicable to any species. Examples of open systems include GeneCalling^®, SAGE, TOGA, READS™, and their progenitor DGE technologies, differential display and cDNA representational difference analysis. We review these technologies and summarize a specific application using GeneCalling for novel gene discovery. Additionally, the significance of data management and experimental design in this new age of expression analysis is discussed.     

PPV long-distance movement is occasionally permitted in resistant apricot hosts

The interactions between Plum pox virus (PPV), a member of the Potyvirus genus, and Prunus host plants are, up to now, poorly understood. In the current paper, fluorescence stereomicroscopy, in situ hybridisation and immunogold detection were performed in order to evaluate the virus transport and cellular distribution. The behavior of PPV in several susceptible (cv. ‘Moniqui’ and ‘Screara’) and resistant apricot genotypes (cv. ‘Harlayne’, ‘Henderson’, ‘Harcot’, ‘Goldrich’, ‘Stella’ and ‘Stark Early Orange’) were compared. Viral RNA was detected by in situ hybridisation in stem tissues close to the inoculation point, irrespective of the resistance status of the variety. Systemic infection was evidenced by virus immunodetection and by fluorescence detection of a GFP-tagged PPV in distant leaf sections. The signal obtained by in situ hybridisation colocalised with the fluorescence produced by GFP-tagged PPV in the same plant material but did not colocalise with the signal obtained by immunostaining. Intensity of the PPV infection in susceptible apricot cultivars varied depending on genotypes. The behavior of PPV in systemic leaves was clearly distinct between susceptible and resistant cultivars. While PPV was spreading widely around the major and minor veins in susceptible leaves, in the resistant apricot genotypes it was restricted to isolated spots consisting of few cells embedded in the mesophyll tissue. In summary, differences in the ability of PPV to systemically infect susceptible and resistant apricot cultivars were evident but nevertheless, long-distance transport of PPV occured in resistant apricot scions.     

Heart rate response is longer after negative emotions than after positive emotions

Summary. Recent ambulatory findings showing comparable cardiovascular effects of positive and negative emotions are not consistent with the assumed etiological role of negative affect in stress-related diseases. We tested the hypothesis that regardless of initial reactivity, responses associated with negative emotions would be prolonged compared to responses associated with positive emotions. During 8 h, 33 healthy subjects from a general population reported their emotional arousal, emotional valence and physical activity and recorded their heart rates (HR) after a beep at each 60th min (‘initial HR’; T0), followed by two ‘prolonged activation’ recordings, respectively 5 min later (T1) and 10 min later (T2). While emotional arousal and activity predicted initial HR, prolonged activation at T1 was solely predicted by emotional valence (negative affect) at T0, independent of emotional recovery. The results lend support to the hypothesis that cardiovascular activation after negative emotions last longer than after positive emotions. This finding is consistent with the view that prolonged activation, and not so much reactivity, might be a mechanism underlying the etiological role of negative emotions (‘stress’) in somatic disease. Perseverative cognition (worry, rumination) might be responsible for this prolonged activation.     

Preprocessing histograms of age at menopause using the fast Fourier transform

Objective: In epidemiological and biological studies, the interpretation of frequency distributions (histograms) of the age at menopause (AAM) is hampered by the presence of background noise such as the last-digit-preference (LDP) of each woman. The objective of this study was to develop a standard method of preprocessing the AAM histogram such that noise can be effectively eliminated, thereby enabling a more thorough investigation of the underlying properties of the distribution. Methods: The Fast Fourier transform (FFT) is a technique which eliminates individual sources of noise based on their characteristic frequencies. Its effectiveness in eliminating noise from AAM data was explored using both simulated and published data, especially in regard to the elimination of cyclical LDP errors. Results: By preprocessing the histogram ‘signal’ using a low-pass filter of 0.15 cycles per year (cpy), common LDP noise is eliminated. Furthermore, this preprocessing also eliminates other forms of high-frequency noise, revealing a true AAM ‘signal’ which comprises three separate low-frequency components. The two major peaks correspond in the time domain to Gaussian functions with means at 51 and 43 years and peak-widths of 6 years; a smaller peak also exists at 35 years. Conclusions: The FFT is an effective tool in preprocessing AAM frequency distributions to reveal their underlying shape, which appears similar across several previously published studies and is characterized by three distinct peaks. These currently have no definitive interpretation, but call into question the analysis of epidemiological risk factors for AAM using statistical techniques which assume a single distribution.     

Understanding the physiology and adaptation of staphylococci: a post-genomic approach

Staphylococcus aureus as well as coagulase-negative staphylococci are medically highly important pathogens characterized by an increasing resistance rate toward many antibiotics. Although normally being skin and mucosa commensals, some staphylococcal species and strains have the capacity to cause a wide range of infectious diseases. Many of these infections affect immunocompromised patients in hospitals. However, community-acquired staphylococcal infections due to resistant strains are also currently on the rise. In the light of this development, there is an urgent need for novel anti-staphylococcal therapeutic and prevention strategies for which a better understanding of the physiology of these bacteria is an essential prerequisite. Within the past years, staphylococci have been in the focus of genomic research, resulting in the determination and publication of a range of full-genome sequences of different staphylococcal species and strains which provided the basis for the design and application of DNA microarrays and other genomic tools. Here we summarize the results of the project group ‘Staphylococci’ within the research network ‘Pathogenomics’ giving new insights into the genome structure, molecular epidemiology, physiology, and genetic adaptation of both S. aureus and coagulase-negative staphylococci.     

Opinion survey towards hormone replacement therapy in the prevention of coronary heart disease

Background: Observational and experimental data underscore the cardioprotective effects of hormone replacement therapy (HRT). On the other hand, the randomised trial available, the ‘Heart and Estrogen/Progestin Replacement Study (HERS)’, showed no reduced risk of coronary heart disease (CHD), using HRT. Aim: Opinion survey on the effect of HRT on CHD risk. Setting: Identification of articles on the related topic using a Medline search. Written survey of the authors’ opinion towards HRT in relation to CHD. Results: Thirty-seven of the 108 principal authors responded. Among them, respectively, 16 (43%) and seven (19%) found that HRT has favourable effects on primary and secondary prevention, two (5%) and five (14%) that it had no effect, none (0%) and four ( 11%) that it had an unfavourable effect, seven (19%) and nine (25%) that it had both favourable and unfavourable effects, and nine (25%) and ten (27%), thought that there are not enough data. Considering a risk modification superior to 20% as clinically relevant, then 57% thought that HRT has a beneficial effect of on primary prevention and 30% on secondary prevention, while none of the responders considered that HRT has unfavourable effects on primary prevention and only 2% on secondary prevention of CHD. Conclusion: Despite the negative results of the HERS study, about one-half of the responders still think that HRT has a beneficial effect on primary prevention of CHD and almost one-third on secondary prevention.     

The effect of alcohol on blood glucose in Type 1 diabetes--metabolic modelling and integration in a decision support system

Introduction: We have recently shown, in studies with patients with Type 1 (insulin dependent) diabetes, that alcohol intake at 21:00 h significantly reduced blood glucose values after 10–12 h, compared with control studies with no alcohol. Hypothesis: We hypothesised that this was due to the following effects of alcohol: (1) alcohol metabolism increases NADH, leading to a reduction in hepatic gluconeogenesis; (2) increased glycogen phosphorylase activity depletes hepatic glycogen stores; (3) after the alcohol is metabolised, hepatic insulin sensitivity is increased, leading to the restoration of glycogen stores and reduction in blood glucose levels; and (4) consequently, after several hours, glycogen stores and insulin sensitivity return to normal. Results: A model describing these changes (DiasNet-Alcohol) was implemented into the DiasNet model of human glucose metabolism. Our study suggests that the DiasNet-Alcohol model gives a reasonable approximation of these effects of alcohol on blood glucose concentration observed in our study and supports our hypothesis for the mechanism behind these effects in Type 1 diabetes.     

Expression site activation in Trypanosoma brucei with three marked variant surface glycoprotein gene expression sites

The genes for the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei are transcribed in telomeric expression sites (ESs). There are about 20 different ESs per trypanosome nucleus. Usually, only one is active at a time, but trypanosomes can switch the ES that is active at a low rate (<10⁻⁵ per cell per generation). To study activation and silencing of ESs, we have generated a line of T. brucei 427 with three ESs marked with a different drug resistance gene. We show that a selection with any combination of two of these drugs leads to an unstable double-resistant phenotype in which the two ESs containing the corresponding marker genes switch backward and forward at a very high rate (>10⁻¹ per cell per generation). Unstable triple-resistant trypanosomes were not obtained. We conclude that the unstable rapid-switching state is a natural intermediate in ES switching. It only involves two ESs, whereas the other ESs are not expressed. Furthermore, we show that ‘inactive’ ESs can exist at several different stable levels of activation. Whereas, a ‘silent’ ES shows a low level of expression of promoter proximal sequences, the level of activation can be reversibly increased, leading to partially activated ESs.