Sialic acid in erythrocytes of patients with amaurotic idiocy and Huntington's chorea

Summary Total erythrocyte sialic acid and lipid-extractable sialic acid were estimated in material obtained from 14 normal subjects, and from 8 parents and 1 sibling of patients suffering from late infantile or juvenile forms of amaurotic idiocy, from 1 patient in the terminal phase of the disease and 2 patients with Huntington's chorea. In contrast to a previous report no increase in lipid-sialic acid was found, nor were the total sialic acid values in the pathological material raised. The cause of these different results is still obscure. The possible role of lipid-peroxidation products is discussed.

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Zusammenfassung Der Gehalt der Erythrocyten an gesamter N-Acetyl-Neuraminsäure und an Lipid-extrahierbarer N-Acetyl-Neuraminsäure wurde bestimmt bei 14 Normalpersonen sowie 8 Eltern und einem Geschwister von Patienten, die an der spätinfantilen oder juvenilen Form der amaurotischen Idiotie litten — bei einem Patienten im terminalen Stadium der Krankheit und bei zwei Patienten mit Chorea Huntington. Im Gegensatz zu einem früheren Bericht fand sich keine Erhöhung der Lipid-N-Acetyl-Neuraminsäure, und auch der Gesamtwert war nicht erhöht. Die Ursache dieser Diskrepanz der Ergebnisse ist noch unbekannt. Die mögliche Bedeutung von Lipid-Peroxidationsprodukten wird diskutiert.

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This work has been made possible by a subsidy received from the Organization for Health Research T. N. O., The Hague, Netherlands.     

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.     

Genomic structure and evolution of the human pepsinogen A multigene family

Summary A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0–16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.     

Processing of the electroencephalogram in cardiac surgery

The objective of this investigation was to find a method to automatically detect several types of abnormal EEG patterns which occurred during cardiac surgery. The EEGs were analyzed by means of several EEG processing and pattern recognition methods. It was found that a good classification into normal and abnormal EEG patterns was generally obtained if only two EEG features were used. The results of this investigation are of importance for implementing into a computer-based monitoring system for use during cardiac surgery.     

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h⁻¹) was fitted with a 0.22-μm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h⁻¹ (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g⁻¹ of biomass h⁻¹ calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h⁻¹. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.Laboratory studies on microorganisms are often performed in batch cultures. During the initial phase of batch cultivation, all nutrients are usually present in excess. As a consequence, the initial specific growth rate, μ, of the microorganism in such cultures equals the maximum specific growth rate, μ_max. In natural environments, the specific growth rate of microorganisms is likely to be constrained by the limited availability of one or more growth-limiting nutrients, resulting in specific growth rates far below μ_max (8, 24). In chemostat cultures fed with a medium containing a single growth-limiting nutrient, the dilution rate determines the specific growth rate. Chemostat cultivation therefore offers the possibility to study microbial physiology at carefully controlled, submaximal specific growth rates and to investigate the effect of specific growth rate on cellular physiology (20). Chemostat cultivation of the yeast Saccharomyces cerevisiae has demonstrated strong effects of specific growth rate on biomass composition (26, 51), product formation (5, 37), and cell size (23). Moreover, during energy-limited growth at low specific growth rates, a relatively large fraction of the energy substrate has to be dissimilated for maintenance-related processes such as maintenance of chemi-osmotic gradients and turnover of cellular components (34). Not surprisingly, recent genome-wide studies have shown strong effects of specific growth rate on levels of mRNAs and proteins (9, 14, 38).In chemostat studies on S. cerevisiae, the steady-state specific growth rate is usually between 0.03 h⁻¹ and 0.40 h⁻¹. While this range is relevant for many industrial applications, there are several incentives to study growth of this yeast at even lower specific growth rates. In many natural environments, growth at a μ of 0.03 h⁻¹, corresponding to a doubling time of 23.1 h, probably still represents extremely fast growth. Furthermore, in industrial applications, S. cerevisiae and other microorganisms can be considered as self-replicating catalysts, and, unless biomass is the desired product, growth can be considered as undesirable by-product formation leading to nonproductive substrate consumption. This problem is further augmented when the excess yeast biomass cannot be valorized because it is genetically modified or has been used for the production of compounds that are not compatible with use as, for example, cattle feed. A third incentive for exploring the physiology of S. cerevisiae at near-zero growth rates is related to the increasing interest in this yeast as a systems biology model for human cells (16, 27, 33). At near-zero growth rates, the age of individual yeast cells becomes much higher than can be achieved in conventional batch or chemostat cultures. Studies on extremely slow growth of S. cerevisiae under defined conditions may therefore provide an interesting model for ageing of human cells.Retentostat cultivation, first proposed by Herbert (18), is a modification of chemostat cultivation that has been specifically designed to study microbial physiology at near-zero specific growth rates. In a retentostat, sometimes referred to as recycling fermentor or recyclostat, the growth-limiting energy substrate is fed at a constant rate, and biomass is retained in the fermentor by an internal filter probe connected to the effluent line or by an external filter module. Prolonged retentostat cultivation should, in theory, result in a situation where the specific growth rate becomes zero and where the specific rate of substrate consumption equals the maintenance energy requirement. This situation is fundamentally different from starvation, which involves deterioration of physiological processes, and from resting states typified by spores, which have little or no metabolic activity. Retentostat cultivation has been applied to several bacterial systems including Escherichia coli (11), Paracoccus denitrificans, and Bacillus licheniformis (49) and the autotrophs Nitrosomonas europaea and Nitrobacter winogradskyi (46, 47). These studies demonstrated that the physiology of these prokaryotes at extremely low specific growth rates could not be accurately predicted by a simple extrapolation of results obtained at higher specific growth rates. In particular, near-zero specific growth rates coincided with increased levels of ppGpp (2), which induces the stringent response, a regulatory program that diverts cellular resources from growth to amino acid biosynthesis (10, 21). Furthermore, it was concluded that extremely slow growth led to a reduction of the maintenance energy requirement of prokaryotes. A recent quantitative analysis on cell retention cultures of S. cerevisiae was performed under severely nitrogen-limited growth conditions and used incomplete cell retention (7), which precluded a quantitative comparison with maintenance energy requirements calculated from energy-limited chemostat cultures.The goal of the present study was to quantitatively analyze the physiology of S. cerevisiae at extremely low specific growth rates in glucose-limited retentostat cultures. To this end, an internal filter probe was introduced in the effluent line of standard laboratory chemostat fermentors and used in long-term cultivation runs with complete cell retention. Anaerobic conditions were chosen to facilitate quantification of catabolic fluxes and growth energetics.     

Inhibition of type 2A secretory phospholipase A2 reduces death of cardiomyocytes in acute myocardial infarction

During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A₂ (sPLA₂-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA₂-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA₂-IIA activity and reduced infarct size (reduction 73 ± 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA₂-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA₂-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA₂-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA₂-IIA independent manner. A. van Dijk and P. A. J. Krijnen have contributed equally to the study.