Changes in CSF acetyl- and butyrylcholinesterase activity after long-term treatment with AChE inhibitors in Alzheimer's disease.

OBJECTIVES: To measure cerebrospinal fluid (CSF) activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in patients with Alzheimer's disease (AD) participating in randomized clinical trials from three European centers, before and after long-term treatment with different AChE inhibitors (AChEIs). MATERIALS AND METHODS: Of the 144 patients included in the study, 104 were treated with donepezil, 15 with galantamine, 16 with rivastigmine, and nine with placebo. CSF AChE and BChE activities were measured at baseline and after 1- year treatment. RESULTS: Donepezil and galantamine groups showed a significant increase in CSF AChE activity at follow-up, while no changes for BChE activity were observed; in donepezil group, a positive correlation between plasma concentration and AChE activity was documented. Conversely, in rivastigmine group, a decrease in CSF activity of both enzymes was observed. CSF AChE and BChE activities were not correlated with the clinical outcome in any group considered. CSF biomarkers did not show any change after treatment. CONCLUSIONS: AChEIs differently influence the activity of target enzymes in CSF independent of their pharmacodynamic effects.

Fungal biomass associated with the phyllosphere of bryophytes and vascular plants.

Little is known about the amount of fungal biomass in the phyllosphere of bryophytes compared to higher plants. In this study, fungal biomass associated with the phyllosphere of three bryophytes (Hylocomium splendens, Pleurozium schreberi, Polytrichum commune) and three vascular plants (Avenella flexuosa, Gymnocarpium dryopteris, Vaccinium myrtillus) was investigated using ergosterol content as a proxy for fungal biomass. Phyllosphere fungi accounted for 0.2-4.0 % of the dry mass of moss gametophytes, representing the first estimation of fungal biomass associated with bryophytes. Significantly more fungal biomass was associated with the phyllosphere of bryophytes than co-occurring vascular plants. The ergosterol present in moss gametophytic tissues differed significantly between species, while the ergosterol present in vascular plant leaf tissues did not. The photosynthetic tissues of mosses had less associated fungal biomass than their senescent tissues, and the magnitude of this difference varied in a species-specific manner. The fungal biomass associated with the vascular plants studied varied significantly between localities, while that of mosses did not. The observed differences in phyllosphere community biomass suggest their size could be affected by host anatomical and physiological attributes, including micro-niche availability and chemical host defenses, in addition to abiotic factors like moisture and nutrient availability.

Boreal bog plants: nitrogen sources and uptake of recently deposited nitrogen.

The main goals of this study were to determine the delta15N signature of quantitatively important boreal bog plants as basis for discussing their N sources, and to assess the effects of five different 3 year N treatments (i.e. 0, 5, 10, 20 and 40 kg N ha(-1) year(-1)) on the bog plants and surface peat at different depths (i.e. 0, 5, 10, 20 and 40 cm) by using 15N as tracer. Plants and peat were analyzed for N concentration, 15N natural abundance and 15N at.%. From the results we draw three main conclusions: First, the relative importance of different N sources is species-specific among bog plants. Second, an annual addition of 5 kg N ha(-1) year(-1) was sufficient to significantly increase the N concentration in Sphagnum mosses, liverworts and shallow rooted vascular plants, and an annual addition of 40 kg N ha(-1) year(-1) during 3 years was not sufficient to increase the N concentration in deep rooted plants, although the 15N content increased continuously, indicating a possible longer term effect. Third, an annual addition of 40 kg N ha(-1) year(-1) during 3 years increased the N content in surface peat at depths of 5 and 10 cm, but not at depths of 20 and 40 cm, indicating the capacity of the living Sphagnum mosses and the surface peat to take up deposited N, and thereby function as a filter.

Puromycin aminonucleoside damages the glomerular size barrier with minimal effects on charge density.

Puromycin aminonucleoside (PAN) has been suggested to reduce glomerular charge density, to create large glomerular "leaks," or not to affect the glomerular barrier. Therefore, we analyzed glomerular charge and size selectivity in vivo and in isolated kidneys perfused at 8 degrees C (cIPK) in control and PAN-treated rats. The fractional clearances (theta) for albumin and Ficoll of similar hydrodynamic size were 0.0017 +/- 0.0004 and 0.15 +/- 0.02, respectively, in control cIPKs. Two-pore analysis gave similar results in vivo and in vitro, with small- and large-pore radii of 47-52 and 85-105 A, respectively, in controls. Puromycin increased the number of large pores 40-50 times, the total pore area over diffusion distance decreased by a factor of 25-30, and the small-pore radius increased by 33% (P < 0.001 for all comparisons of size selectivity and theta). The effect of PAN was less dramatic on the estimated wall charge density, which was 73% of that of controls. We conclude that puromycin effectively destroys the glomerular size barrier with minimal effects on charge density.

Effects of filtration rate on the glomerular barrier and clearance of four differently shaped molecules.

The effect of shape on the transglomerular passage of solutes has not been hitherto systematically studied. We perfused isolated rat kidneys to determine the fractional clearances (theta) at various filtration rates for four molecules of different shapes but with similar Stokes-Einstein radii (aSE = 34-36 A). The theta for hyaluronan, bikunin, and Ficoll36 A were 66, 16, and 11%, respectively, at a glomerular filtration rate (GFR) of 0.07 ml x min(-1) x g wet wt(-1) and decreased to 46, 14, and 7%, respectively, on a fivefold increase in GFR. Under the same conditions, theta for albumin increased from 0.15 to 0.74%, and similar behavior was observed for larger Ficolls (aSE >45 A). Pore analysis showed that the "apparent neutral" solute radii of Ficoll, albumin, bikunin, and hyaluronan were 35, 64, 33, and 24 A, respectively, despite similar aSE. In addition, the properties of the glomerular filter changed with increasing GFR and hydrostatic pressure. We conclude that elongated shape, irrespective of size and charge, drastically increases the transglomerular passage of a solute, an effect that is related to its frictional ratio.

A quantitative analysis of the glomerular charge barrier in the rat.

Modifying the ionic strength (I) is a gentle way to alter charge interactions, but it cannot be done for studies of the glomerular sieving of proteins in vivo. We therefore perfused 18 isolated rat kidneys with albumin solutions of different ionic strengths at a low temperature (cIPK) to inhibit tubular uptake and protease activity. Four anionic proteins were studied, namely albumin (Alb), orosomucoid (Oro), ovalbumin (Ova), and anionic horseradish peroxidase (aHRP), together with the neutral polymer Ficoll. With normal ionic strength of the perfusate (152 mM), the fractional clearance (theta) was 0.0018 +/- 0.0003 for Alb, 0.0033 +/- 0.0003 for Oro, 0.090 +/- 0.008 for Ova, and 0.062 +/- 0.002 for aHRP. These theta values were all lower than for Ficoll of similar hydrodynamic size; e.g., theta(Ficoll 36 A) was >20 times higher than theta for albumin. Low ionic strength (34 mM) increased size selectivity as theta for anionic proteins and Ficoll fell, suggesting a reduction in small-pore radius from 44 +/- 0.4 to 41 +/- 0.5 A, P < 0.01. In contrast, low I reduced the charge density of the membrane, omega, to one-quarter of the 20--50 meq/l estimated at normal I. These dynamic changes in omega seem to be due to volume alterations of the charged gel, fluid shifts that easily are accounted for by the changes in electroosmotic pressures. The finding that low ionic strength induces inverse effects on size selectivity and charge density strongly suggests that separate structures of the glomerular wall are responsible for the two properties.

Jac1, a mitochondrial J-type chaperone, is involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae.

A minor Hsp70 chaperone of the mitochondrial matrix of Saccharomyces cerevisiae, Ssq1, is involved in the formation or repair of Fe/S clusters and/or mitochondrial iron metabolism. Here, we report evidence that Jac1, a J-type chaperone of the mitochondrial matrix, is the partner of Ssq1 in this process. Reduced activity of Jac1 results in a decrease in activity of Fe/S containing mitochondrial proteins and an accumulation of iron in mitochondria. Fe/S enzyme activities remain low in both jac1 and ssq1 mutant mitochondria even if normal mitochondrial iron levels are maintained. Therefore, the low activities observed are not solely due to oxidative damage caused by excess iron. Rather, these molecular chaperones likely play a direct role in the normal assembly process of Fe/S clusters.

A gel-membrane model of glomerular charge and size selectivity in series.

We have analyzed glomerular sieving data from humans, rats in vivo, and from isolated perfused rat kidneys (IPK) and present a unifying hypothesis that seems to resolve most of the conflicting results that exist in the literature. Particularly important are the data obtained in the cooled IPK, because they allow a variety of experimental conditions for careful analysis of the glomerular barrier; conditions that never can be obtained in vivo. The data strongly support the classic concept of a negative charge barrier, but separate components seem to be responsible for charge and size selectivity. The new model is composed of a dynamic gel and a more static membrane layer. First, the charged gel structure close to the blood compartment has a charge density of 35-45 meq/l, reducing the concentration of albumin to 5-10% of that in plasma, due to ion-ion interactions. Second, the size-selective structure has numerous functional small pores (radius 45-50 A) and far less frequent large pores (radius 75-115 A), the latter accounting for 1% of the total hydraulic conductance. Both structures are required for the maintenance of an intact glomerular barrier.

Glomerular size and charge selectivity in the rat as revealed by FITC-ficoll and albumin.

The fractional clearances (theta) for FITC-Ficoll and albumin were estimated in isolated perfused rat kidneys in which the tubular activity was inhibited by low temperature (8 degrees C) and/or 10 mM NH(4)Cl. The Ficoll data were analyzed according to a two-pore model giving small and large pore radii of 46 A and 80-87 A, respectively. The estimated negative charge density was 35-45 meq/l at 8 degrees C. Perfusion with erythrocyte-free solutions of kidneys at 37 degrees C reduced glomerular size and charge permselectivity. Thus the large pore fraction of the glomerular filtrate (f(L)) was 1.64% at 37 degrees C compared with 0.94% at 8 degrees C. The theta for albumin was four times higher at 37 degrees C than at 8 degrees C (0.86% vs. 0.19%, respectively). NH(4)Cl caused further irreversible damage to the glomerular barrier. We conclude that there are no deleterious effects on the glomerular barrier of a reduction in temperature from 37 degrees C to 8 degrees C. Therefore our data seem to disprove the hypothesis of low glomerular permselectivity and transtubular uptake of intact albumin and support the classic concept of a highly selective glomerular barrier.

Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine.

The plasma protein orosomucoid (alpha(1)-acid glycoprotein) has previously been shown to constitute a critical component of the capillary barrier. The protein has also been suggested to act as an anti-inflammatory mediator in a diversity of experimental situations. Recently we reported that orosomucoid is synthesized by the microvascular endothelial cells per se. In the present study, the effects of orosomucoid on primary cultures of human umbilical vein endothelial cells (HUVEC) were studied using the Cytosensor microphysiometer. We found that 1) orosomucoid (0.01 g/l) increased the metabolic activity of HUVEC as reflected by the increased acidification rate of +14 +/- 1%; 2) pretreatment with 0.5 mM 8-bromo-cAMP for 20 min markedly and reversibly inhibited the effect of orosomucoid, whereas 8-bromo-cGMP did not; 3) histamine elicited a dose-dependent response that was abolished by pretreatment with either cAMP or cGMP; and finally, 4) pretreatment of HUVEC for 6 min with orosomucoid (0.01 g/l) inhibited the action of histamine. In summary, this is the first report demonstrating that orosomucoid affects human endothelial cells and that it does so by using cAMP as a second messenger. This provides an explanation for previous findings of anti-inflammatory effects of the protein and shows that orosomucoid affects the endothelium during both normal and pathophysiological conditions.

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1.

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

Human endothelial cells produce orosomucoid, an important component of the capillary barrier.

The serum protein orosomucoid (alpha1-acid glycoprotein) is needed to maintain the high capillary permselectivity required for normal homeostasis. It is not known how the protein executes its action, but it seems to contribute to the charge barrier. Moreover, recent studies suggest that the endothelial glycocalyx is essential for the charge barrier. The main site of orosomucoid synthesis is the liver, but we wanted to explore the possibility that orosomucoid was synthesized in endothelial cells. Primary cultures of human microvascular endothelial cells (HMVEC) from dermal tissue were established. Human liver cells were used as positive controls, and total RNA was prepared from both cell types. Reverse transcription-polymerase chain reaction (RT-PCR) was performed and demonstrated orosomucoid expression. After RT-PCR, the identities of the PCR products were confirmed by sequencing. RNase protection assay performed on total RNA from the HMVEC confirmed the results from the RT-PCR, i.e., orosomucoid mRNA is expressed by endothelial cells. Synthesis of orosomucoid in both liver and endothelial cells was demonstrated by immunoprecipitation. In conclusion, endothelial cells normally produce orosomucoid, which is essential for capillary charge selectivity. We suggest that orosomucoid exerts its effect by interacting with other components of the endothelial glycocalyx.

Glomerular charge selectivity for horseradish peroxidase and albumin at low and normal ionic strengths.

The classical concept of a negative glomerular charge barrier has recently been questioned, mainly based on the somewhat high clearance for anionic horseradish peroxidase (HRP). The validity of using anionic HRP can be tested by changing the properties of the charge barrier. A rather unequivocal approach is to reduce the ionic composition of the perfusate and hence increase the Debye length. We determined the glomerular clearance for horseradish peroxidase and serum albumin, using isolated rat kidneys perfused at 8 degrees C to reduce the tubular modification of the primary urine. The perfusate contained trace amounts of the neutral 125I-nHRP and the anionic 131I-aHRP and were otherwise identical except for different ionic strengths, 152 mM and 34 mM, respectively. During control, the fractional clearance (theta) was 0.11 +/- 0.015 for nHRP and 0.045 +/- 0.010 for aHRP, with an average clearance ratio (n/a) of 2.8 +/- 0.24. Low ionic strength reduced theta for aHRP to 0.027 +/- 0.006, giving an increased clearance ratio for HRP of 4.2 +/- 0.44. The existence of a negative charge barrier is supported by the experiments. The result obtained during normal perfusion is compatible with a charge density (omega) of 34 mEq L-1, using a model of homogeneously charged membrane. Low ionic strength perfusion reversibly reduced the concentration of fixed charges to 12 mEq L-1, suggesting an almost threefold increase of the glomerular membrane volume. Thus, the glomerular charge barrier should be regarded to have a dynamic gel structure rather than being a rigid membrane.

The HLA-Dw2 haplotype segregates closely with multiple sclerosis in multiplex families.

Multiple sclerosis (MS) is associated with the HLA class II specificity Dw2, but the importance of its influence has been questioned, since sib-pair analysis has failed to show linkage with this haplotype. However, the use of 'identity by descent' (IBD) analysis may not be ideal, since it does not make use of the facts that (i) the Dw2-haplotype is the only haplotype with a confirmed role in MS, and (ii) it performs its influence in a dominant manner. We have investigated nine Swedish multiplex MS families. In eight of the families, the Dw2 haplotype occurred in MS patients. Within these families, Dw2 was shared by all 17 individuals with MS. In a compilation of 48 published multiplex MS families in which at least one patient carried Dw2, only three of 107 individuals with MS did not carry the Dw2 haplotype. This indicates that the Dw2 haplotype, when present in familial MS, may confer a stronger influence in MS susceptibility than generally recognized.

Nitrate assimilation in coexisting vascular plants in mire and swamp forest habitats in Central Sweden.

In order to monitor the nitrate assimilation capability of mire plants, in vivo current and maximally induced nitrate reductase activity (NRA) were investigated in 14 species of vascular plants from four different sites in a central Swedish mire. One of the sites was a swamp forest. The plants studied included species with both wide and restricted ecological ranges, and the mire sites selected covered a wide range of plant productivity. At the most productive site, current NRA differed among coexisting species. This differentiation in the use of nitrate as a source of nitrogen suggested the possibility of resource partitioning with regard to nitrogen acquisition. Maximally induced NRA, measured 3 days after an addition of nitrate, was highest at the most productive sites and differed among coexisting species. Plant species characteristic of rich fens had the highest maximally induced NRA. In all species, there was a positive correlation between the ability to assimilate peaks of available nitrate and total leaf nitrogen concentration.

Longitudinal studies of food intake and weight of women from ages 18 to 56 years.

One hundred fifty-eight women have been studied over a thirty-seven-year period, starting with personal contacts during college and continuing by intermittent mail questionnaires through 1973. Food intakes were calculated from 24-hr. dietary recalls at each sampling. The dietary adjustments in calories and ten nutrients which were made during thirty-seven years were presented. Changes in weight are discussed in relation to dietary changes.

Diet therapy in the U.S. in the past 200 years. A Bicentennial study.

Although diet therapy is a concept of the twentieth century, its foundations were laid by such men as Sanctorius in the sixteenth century, Lavoisier in the eighteenth century, and Beaumont in the nineteenth century, whose detailed notes reflected amazingly accurate observations. With the advent of scientific medicine, research provided the knowledge on which diet therapy was built. Data on food composition, which began to be available around the turn of the century, was important to the therapeutic dietitian, and, at mid-century, formed the basis for the development of the first Exchange Lists (only revised this year). Diets early in the century involved rigid routines, in contrast with the trend today to consider the individual. World War I marked the emergence of the trained dietitian and changes in diet therapy, as knowledge of the biologic sciences and the practice of medium expanded. Research on metabolism led to control of hemoglobin and the red cell anemias, while growing knowledge of the role of pancreatic secretions in metabolism made near-normal lives possible for those with diabetes. The dietitian today finds herself in the position of interpreter of scientific findings, developing meal patterns which not only correct poor food habits but are acceptable to patients. More recently, she has been concerned with problems in modification of fat intake in the interest of possible prevention of cardiovascular disease. Obesity and its prevention remain problems. The practice of diet therapy is subject to vogues, as is science, but the challenge to the dietitian remains: that of serving each patient through the best possible use of her education, skill, and sensitivity.