A regulatory link between ER-associated protein degradation and the unfolded-protein response.

Ubiquitin conjugation during endoplasmic-reticulum-associated degradation (ERAD) depends on the activity of Ubc7. Here we show that Ubc1 acts as a further ubiquitin-conjugating enzyme in this pathway. Absence of both enzymes results in marked stabilization of an ERAD substrate and induction of the unfolded-protein response (UPR). Furthermore, basic ERAD activity is sufficient to eliminate unfolded proteins under normal conditions. However, when stress is applied, the UPR is required to increase ERAD activity. We thus demonstrate, for the first time, a regulatory loop between ERAD and the UPR, which is essential for normal growth of yeast cells.

The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane.

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.

Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins.

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co-purify in successive chromatographic fractionations and co-immunoprecipitated with each other. Tim9p can be cross-linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser67-->Cys67 mutation in Tim9p suppresses the temperature-sensitive growth defect of tim10-1 and tim12-1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.

Diatomaceous earth lowers blood cholesterol concentrations.

In this study a potential influence of diatomaceus earth to lower blood cholesterol was investigated. During 12 weeks we monitored serum lipid concentrations in 19 healthy individuals with a history of moderate hypercholesterinemia (9 females, 10 males, aged 35 - 67 years). Individuals administered orally 250 mg diatomaceous earth three-times daily during an 8 weeks observation period. Serum concentrations of cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglycerides levels were measured before study entry, every second week during the period of diatomaceous earth intake and 4 weeks after stop of intake. Compared to baseline (285.8 +/- 37.5 mg/dl = 7.40 +/- 0.97 mM) diatomaceous earth intake was associated with a significant reduction of serum cholesterol at any time point, reaching a minimum on week 6 (248.1 mg/dl = 6.43 mM, -13.2% from baseline; p<0.001). Also low-density lipoprotein cholesterol (week 4: p<0.05) and triglycerides levels decreased (week 2: p<0.05, week 4: p<0.01). Four weeks after intake of diatomaceous earth was stopped, serum cholesterol, low-density lipoprotein cholesterol and triglycerides still remained low and also the increase of high-density lipoprotein cholesterol became significant (p<0.05). Diatomaceous earth, a bioproduct, is capable of reducing blood cholesterol and positively influencing lipid metabolism in humans. Placebo-controlled studies will be necessary to confirm our findings.

Import of mitochondrial carriers mediated by essential proteins of the intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

A soluble 12-kDa protein of the mitochondrial intermembrane space, Mrs11p, is essential for mitochondrial biogenesis and viability of yeast cells.

We have isolated an essential yeast gene termed MRS11, which codes for a soluble protein of the mitochondrial intermembrane space. Interestingly, this new gene shares many similarities with the previously characterized MRS5 gene: when expressed from a multicopy plasmid, MRS11 like MRS5 restores respiration competence to yeast strains defective in the splicing of mitochondrial group II introns. Both genes are essential for viability of yeast cells, as the disruption of either of them is lethal. The proteins encoded by MRS5 and MRS11, which display 35%, sequence identity are both located in the mitochondrial intermembrane space. Depletion of Mrs11p results in a phenotype similar to that observed in Mrs5p-depleted cells: accumulation of the precursor form of mitochondrial hsp60, inability to form spectrophotometrically detectable amounts of cytochromes and changes in the mitochondrial morphology. Although similar in sequence and function, Mrs5p and Mrs11p are not functionally equivalent and neither can substitute for the other, even when overexpressed. Taken together, our data suggest a cooperative mode of action of Mrs11p and Mrs5p in mitochondrial protein import or other related essential mitochondrial processes.

The yeast ORF YDL202w codes for the mitochondrial ribosomal protein YmL11.

The Saccharomyces cerevisiae open reading frame YDL202w has been characterised in the course of the EUROFAN yeast genome analysis program. Disruption of YDL202w causes a respiratory deficient phenotype accompanied by a loss of mitochondrial DNA. This phenotype is usually found in mutants defective in mitochondrial replication or gene expression. YDL202w has the potential to encode a soluble protein of 249 amino acids. It shows significant similarities to the ribosomal protein L10 from various bacteria and to a previously determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal protein L11. The predicted amino-acid sequence of YDL202w starts with a stretch which has neither any correspondence in the bacterial sequences nor in the protein isolated from mitochondrial ribosomes. Furthermore, this stretch matches the requirements for a signal sequence for mitochondrial protein import. A mitochondrial location of the YDL202w gene product was proven by use of a carboxy terminally HA-tagged version. These findings clearly indicate that YDL202w encodes this mitochondrial ribosomal protein (YmL11).

Properties of low density lipoproteins relevant to oxidative modifications change paradoxically during aging.

Atherosclerosis is a common problem among the elderly. Because lipid peroxidation is considered a contributor to the development of atherosclerosis, we compared oxidative properties of lipoproteins in an otherwise healthy (SENIEUR-classified) aged population (65-74 years) with young controls (18-30 years). Relative amounts of oxidatively altered low density lipoprotein (LDL), estimated by means of an antibody against LDL modified by 4-hydroxynonenal, a product of lipid peroxidation, were increased marginally in serum from the elderly (9.8 vs. 7.4%, P = 0.07). In contrast, isolated LDL from the elderly revealed a decreased susceptibility to in vitro oxidation: the lag time was increased (2.34 vs. 2.10 h, P < 0.01), and the maximal rate of LDL oxidation decreased (0.88 vs. 1.01 O.D./h, P = 0.001). However, there were no age-related changes in lipid composition of native LDL and consumption of fatty acids during in vitro oxidation. The serum concentrations of ascorbic acid and most lipophilic anti-oxidants (the latter expressed per g serum lipids) were significantly decreased in the elderly except tocopherols which tended to be higher. In conclusion, our data reveal paradox age-related alterations of LDL as to its behaviour in oxidation in vivo vs. in vitro.

Mrs5p, an essential protein of the mitochondrial intermembrane space, affects protein import into yeast mitochondria.

We have isolated a yeast nuclear gene that suppresses the previously described respiration-deficient mrs2-1 mutation when present on a multicopy plasmid. Elevated gene dosage of this new gene, termed MRS5, suppresses also the pet phenotype of a mitochondrial splicing-deficient group II intron mutation M1301. The MRS5 gene product, a 13-kDa protein of low abundance, shows no similarity to other known proteins and is associated with the inner mitochondrial membrane, protruding into the intermembrane space. MRS5 codes for an essential protein, as the disruption of this gene is lethal even during growth on fermentable carbon sources. Thus, the Mrs5 protein seems to be involved in mitochondrial key functions aside from oxidative energy conservation, which is dispensable in fermenting yeast cells. Depletion of Mrs5p in yeast cells causes accumulation of unprocessed precursors of the mitochondrial hsp60 protein and defects in all cytochrome complexes. These findings suggest an essential role of Mrs5p in mitochondrial biogenesis.

Molecular characterization of the PEL1 gene encoding a putative phosphatidylserine synthase.

In the yeast Saccharomyces cerevisiae the PEL1 gene is essential for the viability of rho-/rhoo petite mutants, and its mutation in respiring cells results in a pleiotropic phenotype. Results of complementation analysis with different subclones of chromosomal DNA and re-sequencing of the YCL4w-YCL3w segment of chromsome III demonstrate that the coding region of the PEL1 gene corresponds to 1467 bp. The size of the PEL1 transcript in Northern blot analysis was estimated to be approximately 1.5 kb. Transcription initiation in wild-type cells was found to occur at the position -9 relative to the ATG. The PEL1 gene was moderately expressed irrespective of the state of the mitochondrial genome and the nature of the carbon sources. Disruption of the PEL1 gene was not lethal and resulted in the same phenotype as observed with the pel1 mutant, i.e. the cells were not able to survive ethidium bromide mutagenesis, were thermosensitive for growth on glucose at 37 degrees C and failed to grow on minimal glycerol medium. Although the Pel1 protein exhibits significant similarity to a family of phosphatidylserine synthases, the disrupted PEL1 gene was not complemented by the multicopy plasmid-borne CHO1 gene encoding an essential yeast phosphatidylserine synthase.

Lipoprotein(a) and asymptomatic carotid artery disease. Evidence of a prominent role in the evolution of advanced carotid plaques: the Bruneck Study.

BACKGROUND AND PURPOSE: Elevated levels of lipoprotein(a) [Lp(a)] have been reported in association with symptomatic coronary and carotid artery disease. Relevancy of Lp(a) as a risk predictor of presymptomatic atherosclerosis in general populations is not well established. METHODS: Serum Lp(a) distribution and its relation to sonographically assessed carotid atherosclerosis were examined in a random sample of 885 men and women aged 40 to 79 years (Bruneck Study). RESULTS: Logistic regression analysis revealed a binary-type association between Lp(a) and carotid artery disease, with the threshold level of Lp(a) for an enhanced atherosclerosis risk defined at 32 mg/dL. The strength of relation increased with advancing severity of carotid atherosclerosis (odds ratios for Lp(a), 1.8 for nonstenotic and 4.7 for stenotic carotid artery disease; P < .001). Lp(a) was unaffected by environmental factors except for a significant decrease in women taking hormone replacement therapy (P < .05). In a multivariate approach, Lp(a) turned out to be an independently significant predictor of carotid atherosclerosis (P < .001). No differential effect of Lp(a) on atherosclerosis (effect modification) was observed for sex, age, low-density lipoprotein cholesterol, apolipoprotein A-I and B, fasting glucose, diabetes, or hypertension. However, the Lp(a)-atherosclerosis relation was significantly modified by fibrinogen (P < .01) and antithrombin III (P < .05). CONCLUSIONS: The present study demonstrates a strong and independent association between elevated Lp(a) levels and carotid atherosclerosis in a large randomized population and provides evidence of a potential role of Lp(a) in the evolution of carotid stenosis. Apart from atherogenicity of Lp(a) cholesterol, interference with fibrinolysis of atheroma-associated clots and fibrin deposits in the arterial wall may achieve pathophysiological significance.

In vivo LDL receptor and HMG-CoA reductase regulation in human lymphocytes and its alterations during aging.

The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase-polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (P = .030) and HMG-CoA reductase mRNA (P = .062). The age-related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo.

Body iron stores and presence of carotid atherosclerosis. Results from the Bruneck Study.

We hypothesized that the formation of foam cells and fatty streaks requires a postsecretory oxidative modification of lipoproteins that targets them for rapid uptake by macrophages. Lipid peroxidation may in part depend on the concentration of tissue iron, one of the major oxidants in vivo. We analyzed the relation between sonographically assessed carotid atherosclerosis and body iron stores in a population sample of 847 men and women aged 40 to 79 years. In a logistic regression analysis adjusting for age, sex, and all major vascular risk markers, ferritin emerged as one of the strongest indicators of carotid artery disease in both sexes (40 to 59 years; odds ratio, 1.54 per 100 micrograms/L; P < .001). The predictive significance of ferritin was found to be synergistic with that of hypercholesterolemia. Variations in body iron stores between sexes may partly explain evident sex differences in the expression of carotid atherosclerosis. In the elderly (> or = 60 years) the predictive significance of ferritin was found to decrease parallel to that of apolipoprotein B. The current study suggests a possible role of body iron in early atherogenesis.

Increased concentrations of neopterin in carotid atherosclerosis.

Activation of T-cells and macrophages may play a role in the pathogenesis of atherosclerosis. Therefore, serum concentrations of the immune activation markers neopterin and soluble interleukin-2 receptor were compared with routine laboratory parameters, candidate risk variables and degree of carotid atherosclerosis. Study subjects were 561 individuals (293 men and 268 women) aged between 50 and 79 years who were enrolled in a cross-sectional community based study (Ischemic Heart Disease and Stroke Prevention Study, Bruneck, Italy). Extent of carotid atherosclerosis was quantitated by an ultrasound B-mode procedure based scoring system. Detailed physical examination and quantification of laboratory and candidate risk variables were performed. By univariate as well as multivariate statistical analyses, serum concentrations of neopterin but not soluble interleukin-2 receptor were significantly higher in subjects with carotid atherosclerosis (men, 8.5 +/- 2.7 nmol/l neopterin; women, 9.6 +/- 3.3) than in those without (men, 6.7 +/- 2.3, P < 0.0001; women, 7.5 +/- 2.3, P < 0.0001). The data show that the macrophage-derived immune activation marker neopterin is closely correlated with the extent of carotid atherosclerosis. Chronic activation of immune cells, preferentially of macrophages, may play a key role in atherogenesis and/or progression of atherosclerosis.

Estimation of reference intervals from a SENIEUR protocol compatible aged population for immunogerontological studies.

Because many laboratory values change with age, the study of healthy aging as well as diagnosis of disease in geriatric patients requires specific age-corrected reference intervals. We have established such reference intervals for a healthy population aged 65-74 years by selection of a sample group applying the clinical criteria of the SENIEUR protocol and we have compared them with those of a young control group (20-33 years) fulfilling the same criteria. Significant or minor elevations were seen, e.g. for plasma concentrations of fasting glucose, urea, total and LDL-cholesterol, triglycerides, gamma-glutamyl-transferase, alkaline phosphatase, erythrocyte sedimentation rate and serum neopterin levels. These reference intervals can be used for selecting a SENIEUR compatible population aged between 65 and 74 years. Additionally, plasma lipid parameters (cholesterol, triglycerides) are proposed to be included in the SENIEUR protocol.

Caries susceptibility and renal excretion of calcium.

Clearance studies were performed for 2 days in two groups of age-matched young female volunteers: those with low caries prevalence and those with high caries prevalence. Both groups were kept on a low-calcium diet for 1 week and received 0.5 g calcium at the beginning of the second day. In both groups, glomerular filtration rate, urinary flow rate and renal excretions of sodium, calcium, and phosphate were subject to significant circadian variations. In both groups the administration of calcium led to a significant increase in renal excretion of sodium and calcium and a significant decrease in that of phosphate. On the first day, calcium excretion was significantly greater in those with low caries prevalence than in those with high caries prevalence, pointing to altered calcium homeostasis in this group.

[Beta-endorphins during childbirth under transcutaneous electric nerve stimulation]. / Verhalten von Beta-Endorphin während der Geburt unter transkutaner elektrischer Nervenstimulation.

Beta-endorphins are substances produced by the body to inhibit the perception of pain. Normally they exhibit a steep rise during birth. We have seen an impressive fall which also was statistically highly significant after the application of transcutaneous electric nerve stimulation in 20 parturient women.

[The automated determination of the dibucaine number using the Greiner G450 selective analyzer. A routine parameter of significance?]. / Die automatisierte Bestimmung der Dibucainzahl am selektiven Analyzer GREINER G450. Ein Routineparameter von Bedeutung?

UNLABELLED: Patients who are homozygous for an atypical pseudocholinesterase enzyme (PCHE) suffer a prolonged neuromuscular block after succinylcholine application. In order to determine which patients have atypical PCHE preoperatively, an automated method using the Greiner G450 analyzer was developed. PATIENTS AND METHODS: The contribution of blocked and unblocked PCHE by dibucaine hydrochloride (optimal concentration 10(-4) mol/l;) was determined in 113 patients (ASA groups 1-2) and the dibucaine number (DN) was evaluated. RESULTS: According to the DN, the patients were subdivided into three groups: group A (PCHE 5.01 +/- 1.64 kU/l, DN 74.47 +/- 0.87); group B (PCHE 4.28 +/- 3.41 kU/l, DN 64.95 +/- 3.41); group C (PCHE 1.33 +/- 0.54 kU/l, DN 13.08 +/- 2.19;). PCHE and DN of group A corresponded with normal standard values, whereas the patients in groups B and C could be considered to be patients with heterozygous and homozygous atypical PCHE, respectively. CONCLUSIONS: Our data indicate that an automated analysis of blocked and unblocked PCHE with the Greiner G450 can be easily done in a routine laboratory. By interpreting the DN, the possible risks of delayed succinylcholine degradation can probably be prevented.