CAP2, cyclase-associated protein 2, is a dual compartment protein.

Cyclase-associated proteins (CAPs) are evolutionarily conserved proteins with roles in regulating the actin cytoskeleton and in signal transduction. Mammals have two CAP genes encoding the related CAP1 and CAP2. We studied the distribution and subcellular localization of CAP1 and CAP2 using specific antibodies. CAP1 shows a broad tissue distribution, whereas CAP2 is significantly expressed only in brain, heart and skeletal muscle, and skin. CAP2 is found in the nucleus in undifferentiated myoblasts and at the M-line of differentiated myotubes. In PAM212, a mouse keratinocyte cell line, CAP2 is enriched in the nucleus, and sparse in the cytosol. By contrast, CAP1 localizes to the cytoplasm in PAM212 cells. In human skin, CAP2 is present in all living layers of the epidermis localizing to the nuclei and the cell periphery. In in vitro studies, a C-terminal fragment of CAP2 interacts with actin, indicating that CAP2 has the capacity to bind to actin.

A thirteen week dietary toxicity study with 7-hydroxymatairesinol potassium acetate (HMRlignan) in rats.

Plant lignan 7-hydroxymatairesinol (7-HMR) is a novel precursor of the mammalian lignan enterolactone. A 13 week toxicity study at dietary levels of 0, 0.25, 1, and 4% (w/w) of potassium acetate complex of 7-HMR (HMRlignan) was conducted in the Wistar rat. These dietary levels resulted in an average daily intake of 160, 640, and 2600 mg HMRlignan/kg body weight/day, respectively. A considerable systemic exposure of HMRlignan was verified by dose-related increases in plasma total (conjugated and unconjugated) concentration of 7-HMR and metabolites enterolactone, 7-hydroxyenterolactone, and matairesinol. Enterolactone appeared to be the major metabolite. Most (>96%) of the circulating 7-HMR and enterolactone was in conjugated form as measured from the low-dose rat plasma samples. HMRlignan exposure did not significantly affect clinical signs, ophthalmoscopy or neurobehavioural observations, and motor activity. Transient reductions in food intake and body weight gain in the mid-and high-dose group were ascribed to decreased palatability of the test feed. Only in males of the high-dose group the body weights remained slightly reduced throughout the study. In the high-dose group the number of thrombocytes (females), and total white blood cell count (males) were increased. Plasma triglycerides were dose-dependently depressed in males of all test groups and in females of the mid- and high-dose group, while plasma total cholesterol, and phospholipids were decreased in high-dose males. These changes, which have also been reported for other (flaxseed) lignans, were not considered to represent adverse effects. The relative weight of the kidneys was increased in males of the high-dose group. The weight of the full and empty caecum showed dose-related increases in males of all treatment groups and in females of the high-dose group. Absolute ovary weights were decreased in all treatment groups while decreases in relative ovary weights were confined to the mid- and high-dose group. In addition, a marginal lengthening of the estrus cycle was noted in high-dose females. Apart from prevention of hyaline droplet nephropathy in all high-dose male rats, there were no treatment-related histopathological alterations. It was concluded that HMRlignan showed weak antiestrogen-like activity which may be mediated through enterolactone metabolite. Based on declined ovary weight, the no observed adverse effect level of HMRlignan was set at 0.25% in feed corresponding to 160 mg/kg body weight/day.

Prenatal developmental toxicity study with 7-hydroxymatairesinol potassium acetate (HMRlignan) in rats.

Plant lignan 7-hydromatairesinol, a novel precursor of the mammalian lignan enterolactone was evaluated in a prenatal developmental toxicity study conducted in the Wistar rat. Mated female rats were fed diets containing 0, 0.25, 1, and 4% (w/w) of 7-hydroxymatairesinol in the form of potassium acetate complex (HMRlignan; potassium acetate level approximately 20% w/w within the preparation) for days 0-21 of gestation. Test substance intake was calculated to be 0.14-0.18, 0.46-0.74, and 1.19-2.93 g/kg body weight/day for the low, mid, and high-dose groups, respectively. The rats were sacrificed on day 21 of the gestation period and examined for standard parameters of reproductive performance (fecundity index, gestation index, number of corpora lutea, number of implantations, pre- and post-implantation loss, number of early- and late resorptions, number of live- and dead fetuses, sex-ration and the weight of the reproductive organs). The fetuses were examined for external, visceral, and skeletal alterations. The results from this study showed no effects on reproductive performance or any treatment related findings following external, visceral, and skeletal examination of the fetuses. However, approximately half of the mated dams of the high-dose failed to thrive due to an unexpected large decrease in their food intake, and were sacrificed early. Body weights of the remaining animals of the high-dose group were decreased. Food consumption was decreased in all treatment groups during the first three days of the gestation period as a result of decreased palatability of the feed. In conclusion, the no-observed-effect level (NOEL) for maternal effects was 1%, whereas the NOEL for fetal development following daily oral HMRlignan administration throughout the gestation was equivalent to 4% in the diet.

Sea buckthorn berry oil inhibits platelet aggregation.

A small-scale preliminary cross-over study was conducted to investigate the effects of supercritical CO(2)-extracted sea buckthorn berry oil (SBO) on some risk factors of cardiovascular disease. Special features of the oil are high proportions of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), linoleic (18:2n-6), and alpha-linolenic (18:3n-3) acids as well as vitamin E, carotenoids, and sterols. Twelve healthy normolipidemic men were recruited and each volunteer consumed SBO and fractionated coconut oil (control) 5 g per day for a period of 4 weeks in a random order (wash-out 4-8 weeks). Phospholipid fatty acids, plasma lipids, and glucose were unaffected by SBO supplementation. Instead, a clear decrease in the rate of adenosine-5'-diphosphate-induced platelet aggregation and maximum aggregation were found. This suggested the beneficial effects of SBO on blood clotting, but further studies on the dose-response effects are needed to assess the practical use of SBO supplements.

Effects of light exposure and sleep displacement on dim light melatonin onset.

The purpose of the study was to induce in two different ways, a phase-angle difference between the circadian pacemaker and the imposed sleep-wake cycle in humans, we intended to: (i) shift the circadian pacemaker by exposure to bright light and keep the timing of the sleep-wake cycle fixed; and (ii) keep the timing of the circadian pacemaker fixed by a constant light-dark cycle and displace sleep. We monitored dim light melatonin onset (DLMO), core body temperature and sleep. DLMO was delayed significantly after 3 days of a 3-h delayed sleep-phase when compared with 3 days of sleep at a normal or 3-h advanced sleep-phase. The shifts in DLMO were not accompanied by shifts in body temperature, changes in waking-up time or by a change in the duration of the first rapid eye movement (REM) sleep episode. Three days of light exposure in the morning or evening resulted in shifts in DLMO of similar magnitude, but this was accompanied by shifts in the rhythm of body temperature, changes in waking-up time and in the duration of the first REM sleep episode. We conclude that the changes observed after light exposure reflect shifts in the circadian pacemaker. In contrast, we propose that the changes observed in DLMO after sleep displacement are not mediated by the circadian pacemaker. These results raise some doubts about the reliability of DLMO as a marker of circadian phase in cases of sleep disturbances. Finally, we initiate a search for changes in sleep that might be responsible for the unexpected effects on DLMO.

Testing the hypothesis of a circadian phase disturbance underlying depressive mood in nonseasonal depression.

In a crossover design, 8 nonseasonal depressed subjects, selected on the presence of diurnal mood variations, and 8 sex- and age-matched controls were exposed to dim light (< 10 lux) in the evening (18:00-21:00 h) and bright light (2500 lux) in the morning (ML, 6:00-9:00 h), to dim light in the morning and bright light in the evening (EL), or to dim light both in the evening and in the morning (DL) during 3 consecutive days in each of these conditions. There were no initial phase differences between depressed and healthy subjects in the timing of dim light melatonin onset, sleep termination, and body temperature. The phase shifts after EL and ML in both healthy and depressed subjects were as expected on the basis of a human phase response curve. On average, there was no therapeutic effect of the light exposure in the depressed patients. Two patients improved, but these effects do not seem to be related to shifts in the circadian system.

A longitudinal study of sleep deprivation responses in depression; The variability is highly related to diurnal mood variability.

Unequivocal results demonstrating a causal relationship between a disturbance in circadian rhythms and depression have not yet been reported (reviews). However, acute mood changes, such as the antidepressive effect of sleep deprivation, diurnal variations of mood and their interrelationship, are commonly put forward as evidence of the importance of circadian dysregulations in affective disorders. The purpose of the present study is to obtain more insight in the mechanisms underlying these mood changes. The results will be discussed in the context of a recently postulated non-chronobiological explanation. Earlier studies have suggested that the relationship between diurnal variation of mood and the response to total sleep deprivation (TSD) is clear and unambiguous: improvement of mood during the day prior to TSD (a positive diurnal variation) is followed by a positive response (mood improvement) to TSD, while no improvement or deterioration of mood during the day prior to TSD (a negative diurnal variation) may result in no, or even a negative, TSD response (for references see Van den Hoofdakker). However, these conclusions were based on the results from cross-sectional studies, comparing single TSD effects across individuals. Comparison of sleep deprivation effects within individuals, however, revealed that the course of mood during the day prior to TSD is irrelevant for the TSD response. Accordingly, a favourable response to TSD appeared to be related to the patient's propensity to show diurnal mood variations per se, irrespective of their direction.

Sequence analysis of lantibiotics: chemical derivatization procedures allow a fast access to complete Edman degradation.

Lantibiotics are antibiotic peptides produced via ribosomal synthesis of precursor proteins by gram-positive bacteria. They contain various unusual post-translational modifications, which include the formation of sulfide rings by lanthionine or beta-methyllanthionine, and 2,3-didehydroamino acids. The N-terminus may be blocked by a 2-oxobutyryl group and the C-terminus may be inaccessible in some of the lantibiotics. Due to these modifications the analysis of such peptides is very tedious. Chemical modifications using an ethanethiol-containing reaction mixture and/or trifluoroperacetic acid treatment were used to solve these analytical problems. Investigating the tetracyclic 22-peptide gallidermin and the N-terminally blocked tricyclic 34-peptide Pep5 as examples, a novel access to the primary structure of lantibiotics is demonstrated.

Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors.

Injection of poly(A)+ RNA from rabbit small intestine into Xenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino beta-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H(+)-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in oocytes clamped at a holding potential of -60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

Short report: clarithromycin, an alternative to metronidazole in the triple therapy of Helicobacter pylori infection.

BACKGROUND: Triple therapy for Helicobacter pylori using metronidazole is less effective in patients with a metronidazole resistant strain. Moreover, metronidazole is responsible for many side-effects. This open study examined the efficacy and side-effects of a triple treatment regimen substituting clarithromycin for metronidazole. METHODS: 36 patients with a H. pylori infection, proven by culture, were treated with tripotassium dicitrato bismuthate 120 mg q.d.s., tetracycline 250 mg q.d.s. and clarithromycin 250 mg q.d.s. for 10 days. Eradication was defined as a negative culture and histological examination of antral biopsy specimens, taken at least 6 weeks after completion of the treatment. RESULTS: Eradication was achieved in 26 patients (72%). The treatment was well tolerated with only 4 (11%) of the patients having significant side-effects. CONCLUSION: Triple therapy with clarithromycin seems to be less effective than standard triple treatment when the prevalence of metronidazole resistance is low. It is suggested, however, that this combination could be a valuable alternative in areas with a high prevalence of metronidazole resistance.

Strategies for nonradioactive methods in the localization of phosphorylated amino acids in proteins.

Identification of O-phosphorylated amino acids within the primary structure of regulatory proteins is important in understanding the mechanisms by which their functions are regulated. In many cases radioactive labeling with [32P]phosphate is tedious or sometimes impossible. Therefore, we have established a series of new non-radioactive methods that permit the localization of phosphoserine, phosphothreonine, and phosphotyrosine. After partial hydrolysis of a phosphopeptide or phosphoprotein, phosphoserine, phosphothreonine, or phosphotyrosine are determined by capillary electrophoresis as their dabsyl-derivatives. Chemical modification transforms phosphoserine or phosphothreonine to S-ethyl-cysteine or beta-methyl-S-ethyl-cysteine, respectively, allowing their localization during sequence analysis. We apply solid-phase sequencing to overcome the limitations of the gas-phase sequenator in the case of phosphotyrosine-containing peptides. Liquid chromatography on-line connected to an electrospray mass spectrometer is a powerful new method of increasing importance in the protein chemistry field. It is especially well suited for identification of phosphoserine- or phosphothreonine-containing peptides in a proteolytic digest of a phosphoprotein. In this article we will describe how to work with these new methods practically.

Occurrence of an actin-inserting domain in tensin.

We have previously described a protein called "insertin" that binds strongly to barbed ends of actin filaments and permits polymerization of actin filaments by insertion of actin monomers between the barbed ends and barbed end-bound insertin. We determined the amino acid sequence of insertin and found that the primary structure of insertin is almost identical to amino acid residues 862 to 1212 of the actin-binding protein tensin.

Quantitative determination of phosphoserine by high-performance liquid chromatography as the phenylthiocarbamyl-S-ethylcysteine. Application to picomolar amounts of peptides and proteins.

A method is described that permits the phosphoserine content of proteins and peptides to be determined in picomolar amounts. A micro-batch reaction first converts phosphoserine into S-ethylcysteine. Hydrolysis with 6 M hydrochloric acid then yields the free amino acid, which is coupled with phenyl isothiocyanate to give the corresponding phenylthiocarbamylamino acid. This derivative is determined quantitatively in the range 10-20 pmol by reversed-phase high-performance liquid chromatography. The method works well with either small peptides or proteins in the low picomole range.

Sequence analysis of phosphoserine-containing peptides. Modification for picomolar sensitivity.

Sequencing of phosphoserine-containing peptides yields normally no identifiable PTH-derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one-step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as beta-methylaminoalanine (MAA), S-ethanolcysteine or S-ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas-phase sequencer equipped with an on-line PTH amino acid analyzer.

[Xanthogranulomatous pyelonephritis with cancer of the kidney pelvis--a rare coincidence]. / Xanthogranulomatöse Pyelonephritis mit Nierenbeckenkarzinom--eine seltene Koinzidenz.

Xanthogranulomatous pyelonephritis is a specific form of a chronically destructive inflammation of the kidney. In addition to our own case of the extremely rare coincidence of xanthogranulomatous pyelonephritis and a transitional cell carcinoma of the renal pelvis our report also refers to seven further cases of xanthogranulomatous pyelonephritis. Any preoperative diagnosis usually is inaccurate. The symptoms are: general poor health, renal pain, fever, marked reduction or complete loss of the renal function, and frequently radiological evidence of a renal tumor. Surgery of xanthogranulomatous pyelonephritis often results in nephrectomy.

Comparison of the conventional methods and high-performance liquid chromatography for the determination of trimethoprim, sulfamethoxazole and its metabolite in serum.

A modified high-performance liquid chromatographic (HPLC) method for sensitive and rapid determination of trimethoprim, sulfamethoxazole and its metabolite N4-acetylsulfamethoxazole has been compared with the bioassay for trimethoprim and a colorimetric procedure for sulfonamides. The sensitivity of the (HPLC) method has been increased by ultrafiltration of the sample. Thus, the sample dilution was markedly reduced compared to the values obtained with precipitation procedures. The recovery was 102.7 +/- 6.1% for trimethoprim, 93 +/- 5.4% for sulfamethoxazole and 90.2 +/- 7.9% for N4-acetylsulfamethoxazole. The between-day reproducibility was 5% (n = 5). The coefficients of correlation for HPLC and reference methods were 0.993 (bioassay) and 0.995 (colorimetric assay).

[Folic acid substitution in hemodialyzed patients. Folic acid levels in plasma and erythrocytes]. / Folsäure-Substitution bei Hämodialyse-Patienten. Untersuchungen zum Fols-5aaure-Spiegal in Plasma und Erthrozyten

Investigations of folic acid levels in plasma and erythrocytes. In 20 patients on hemodialysis with and without folic acid substitution the concentration of folic acid in plasma and in red cells was estimated by radioassay. In patients, who were substituted with folic acid concomitantly with elevated folic acid concentrations hematocrit values were higher than those of patients who were not substituted. Folic acid concentrations in patients not substituted. Folic acid concentrations in patients not substituted were subnormal and further decreased on dialysis. Results of folic acid determinations in plasma were paralleled by concentrations in erythrocytes. It is concluded, that folic acid substitution is necessary in patients on hemodialysis.