Generation of two induced pluripotent stem cell lines (HIMRi006-A and HIMRi007-A) from Pompe patients with infantile and late disease onset.

Here we present the generation of HIMRi006-A and HIMRi007-A Pompe disease (PD) patient derived human induced pluripotent stem cell (hiPSC) lines. HIMRi006-A represents an infantile onset disease (IOPD) phenotype caused by a homozygous c.307 T > G mutation in the GAA gene. HIMRi007-A is characterized by heterozygous mutations c.-32-13 T > G/c.1716C > G and is associated with an adult onset of disease symptoms (LOPD). Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4, and c-MYC. The lines display a typical embryonic stem cell morphology, express pluripotency markers, retain a normal karyotype (46, XX/XY) and have the differentiation capacity in all three germ layers. Altogether, both lines provide a resource tool to the community for future in depth molecular studies of PD pathomechanism.

Generation of two hiPSCs lines of two patients carrying truncating mutations in the dimerization domain of filamin C.

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi004-A and HIMRi005-A from dermal fibroblasts of a 48-year-old female (HIMRi004-A) carrying missense mutation that translate to the first described filamin C isoform p.W2710X and from a 56-year-old female (HIMRi005-A) carrying a recently described mutation in the same domain p.Y2704X. Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. The lines display a typical embryonic stem cell-like morphology, express pluripotency markers, retain a normal karyotype (46, XX) and have the differentiation capacity in all three germ layers. The two lines can be used to elucidate the pathomechanisms of FLNC myofibrillar myopathies and to develop novel therapeutic options.

Location and restoration of function after cerebellar tumor removal-a longitudinal study of children and adolescents.

Sequelae in children following cerebellar tumor removal surgery are well defined, and predictors for poor recovery include lesions of the cerebellar nuclei and the inferior vermis. Dynamic reorganization is thought to promote functional recovery in particular within the first year after surgery. Yet, the time course and mechanisms of recovery within this critical time frame are elusive and longitudinal studies are missing. Thus, a group of children and adolescents (n = 12, range 6-17 years) were followed longitudinally after cerebellar surgery and compared to age- and gender-matched controls (n = 11). Patients were examined (1) within the first days, (2) 3 months, and (3) 1 year after surgery. Each time behavioral tests of balance and upper limb motor function, ataxia rating, and a MRI scan were performed. Data were used for subsequent lesion-symptom mapping of cerebellar function. Behavioral improvements continued beyond 3 months, but were not complete in all patients after 1 year. At that time, remaining deficits were mild. Within the first 3 months, cerebellar lesion volumes were notably reduced by vanishing edema. Reduction in edema affecting the deep cerebellar nuclei but not reduction of total cerebellar lesion volume was a major predictor of early functional recovery. Persistent impairment in balance and upper limb function was linked to permanent lesions of the inferior vermis and the deep cerebellar nuclei.

Generation of two human iPSC lines (HIMRi002-A and HIMRi003-A) derived from Caveolinopathy patients with rippling muscle disease.

Here we introduce the human induced pluripotent stem cell lines (hiPSCs), HIMRi002-A and HIMRi003-A, generated from cultured dermal fibroblasts of 61-year-old (HIMRi002-A) and 38-year-old (HIMRi003-A) female patients, carrying a known heterozygous pathogenic variant (p.A46T) in the Caveolin 3 (CAV3) gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi002-A and HIMRi003-A display typical embryonic stem cell-like morphology, carry the p.A46T CAV3 gene mutation, express several pluripotent stem cell markers, retain normal karyotype (46, XX) and can differentiate in all three germ layers. We postulate that the HIMRi002-A and HIMRi003-A iPSC lines can be used for the characterization of CAV3-associated pathomechanisms and for developing new therapeutic options.

Impact of Implementing an Elaborated CT Perfusion Protocol for Aneurysmal SAH on Functional Outcome: CTP Protocol for SAH.

BACKGROUND AND PURPOSE: The acute phase of aneurysmal SAH is characterized by a plethora of impending complications with the potential to worsen patients' outcomes. The aim of this study was to evaluate whether an elaborated CTP-based imaging protocol during the acute aneurysmal SAH phase is able to prevent delayed infarctions and contribute to a better outcome. MATERIALS AND METHODS: In 2012, an elaborated CTP-based protocol was implemented for the management of patients with aneurysmal SAH. Retrospective analysis of patients with aneurysmal SAH treated from 2010 to 2013 was performed, comparing the patients treated before (group one, 2010-2011) with those treated after the protocol implementation (group two, 2012-2013) with regard to delayed infarctions and outcome according to the mRS at 3-months' follow-up. RESULTS: A total of 133 patients were enrolled, of whom 57 were included in group 1, and 76, in group 2. There were no significant differences between the groups concerning baseline characteristics. In the multivariate analysis, independent predictors of a good outcome (mRS ≤ 2) were younger age (P < .001), lower World Federation of Neurosurgical Societies grade (P < .001), absence of delayed infarction (P = .01), and management according to the CTP protocol (P = .01). Larger or multiple infarctions occurred significantly more often in group 1 compared with group 2 (88% versus 33% of all delayed infarctions, P = .03). The outcome in group 2 was significantly better compared with group 1 (P = .005). CONCLUSIONS: The findings suggest that implementation of an elaborated CTP protocol is associated with a better outcome. An earlier initiation of further diagnostics and treatment with prevention of large territorial and/or multiple infarctions might have led to this finding.

Zolmitriptan inhibits neurogenic inflammation and pain during electrical stimulation in human skin.

BACKGROUND: Triptans are agonists to 5-HT 1B/D/F receptors, which are present on nociceptive neurons not only within but also beyond the trigeminal system. The aim of this study was to investigate whether zolmitriptan interacts with peptidergic nociceptive afferents in human skin. METHODS: Twenty participants (13 women, median age: 25; interquartile range: 23-26 years) entered the randomized, double-blind, cross-over study. Electrically induced neurogenic flare and pain was assessed after either placebo or zolmitriptan on the ventral thigh. Mechanical pain thresholds were investigated at baseline and after electrical stimulation at the stimulation site. RESULTS: The size of the neurogenic flare (F = 10.9; p = 0.002) as well as electrically induced pain were significantly reduced by zolmitriptan (F = 4.46; p = 0.041). Moreover, electrically induced pinprick hyperalgesia was significantly decreased by zolmitriptan compared with placebo (F = 6.243; p = 0.017). CONCLUSIONS: Triptans may have effects outside of the trigeminal system and reduce electrically evoked neurogenic inflammation and pain in human skin.

Enhanced internal dynamics of a membrane transport protein during substrate translocation.

Conformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of alpha-helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate-loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.

Flavones. 2. Synthesis and structure-activity relationship of flavodilol and its analogues, a novel class of antihypertensive agents with catecholamine depleting properties.

(3-Phenyl-7-flavonoxy)propanolamines have been shown to exhibit antihypertensive activity in spontaneously hypertensive rats. Although they are structurally similar to classical beta-adrenergic blocking compounds, their activity is not due to inhibition of beta-adrenoceptors. In the present study, a series of simple flavonoxypropanolamines was prepared to further explore the structural requirements for the antihypertensive effect of these compounds. A structure-activity relationship of these derivatives indicates that the position of the oxypropanolamine side chain, the hydroxy group of the side chain, steric bulkiness and length of N substituents, degree of the N-substitution, phenyl group at the 2-position of the chromone nucleus, and substituents of the phenyl group or B ring of the flavone play significant roles in imparting pharmacological effects. In addition, there is a good correlation between the antihypertensive activity and depletion of myocardial norepinephrine. Of these analogues tested, the most effective one was flavodilol. Only the 8-substituted analogue 6 was found to be a beta-antagonist. Flavodilol was chosen for in-depth pharmacological, toxicological, and clinical evaluation.

Orally administered immunosuppressants modify intestinal uptake of nutrients in rabbits.

The effect on intestinal nutrient transport of the immunosuppressive drugs cyclosporin A (CsA), cyclosporin G (CsG), and rapamycin (RAP) was determined in New Zealand white rabbits. Rabbits received oral doses of CsA (20 mg/kg/day), CsG (10 mg/kg/day), or RAP (1 mg/kg/day) for 10 days. Animals receiving RAP had decreased food intake and weight gain compared with controls. This correlated with a decrease in both total ileal weight and corresponding mucosal weight. CsA and CsG administration had no effect on food intake, total weight gain, or intestinal weight. Villus surface area was significantly decreased in all groups as compared with controls. Jejunal uptake of D-glucose as well as 1 medium and 4 long chain fatty acids was not affected by drug administration, while both mucosal-to-serosal and net 3-0-methylglucose fluxes were increased (P < 0.05) in the jejunum by all 3 drugs. In the ileum, the rates of uptake of D-glucose as well as stearic and linoleic acids were increased in animals treated with RAP compared with controls. There was an increase in the ileal values of the maximal transport rate (Vmax) and apparent Michaelis constant (Km*) in RAP, and a fall in the Vmax and Km* in CsG. CsG administration resulted in a decreased cholesterol uptake in both jejunum and ileum, and a decreased D-glucose uptake in the ileum compared with controls. These differences in glucose uptake among groups could not be attributed to variations in body, intestinal, or mucosal weights. It is unlikely that the changes observed in CsA- and CsG-treated animals would have nutritional importance, as these animals gained weight normally. In addition, in these animals the changes mainly occurred in the ileum, not in the jejunum, where most glucose is absorbed, and the associated alterations in the values of the Vmax and Km* would lead to reciprocal changes in the rates of uptake of varying luminal concentrations of glucose. In contrast, these changes are likely to be of more importance in RAP-treated animals, since they failed to gain weight normally. The significance of these findings needs to be established in chronically treated animals.

Combinatorial liquid-phase synthesis of

A new method for the synthesis of [1,4]oxazepin-7-ones from readily available aldehydes and alpha-amino alcohols was developed using the Baylis-Hillman reaction as the key step. To determine the scope and limitations of the method, a mixture library was synthesized from six aldehydes and six alpha-amino alcohols on the soluble polymer support poly(ethylene glycol) 5000 monomethyl ether (MeOPEG) via split synthesis and analyzed by GC-EIMS. Those oxazepines that were formed predominantly were resynthesized in a parallel synthesis and fully characterized. Thus, we have shown that split synthesis on MeOPEG can be an efficient method to rapidly screen the substrate spectrum of a newly developed reaction sequence.

Gradient for D-glucose and linoleic acid uptake along the crypt-villus axis of rabbit jejunal brush border membrane vesicles.

Glucose uptake into jejunal brush border membrane (BBM) varies along the crypt-villus axis (CVA). In the present study, the question was addressed whether uptake of the essential long-chain fatty acid linoleic acid also varies along the CVA. Using agitation techniques, five jejunal enterocyte fractions were sequentially isolated from female New Zealand white rabbits. A sixth and final fraction of lower-villus/crypt cells was obtained by the scraping of the remaining jejunal mucosa. Cell fraction along the CVA was proven histologically, by noting decreasing alkaline phosphatase activities in sequentially isolated fractions, and by demonstrating [3H-methyl]thymidine uptake mainly in the final fraction of the lower villus/crypt cells. BBM vesicles were prepared from the upper, mid- and lower-villus/crypt enterocyte fractions, using differential centrifugation and divalent ion precipitation. D-Glucose uptake into each fraction showed an Na(+)-gradient dependent time-course "overshoot" with linear uptake to 15 s and a subsequent decline to a steady-state plateau. Varying D-glucose concentrations from 50-1000 microM demonstrated saturation kinetics of uptake, with maximal transport rates (Vmax) and Michaelis affinity constants (Km) varying between fractions; the Km and Vmax were both lowest in the upper-villus fraction. A linear relationship existed between linoleic acid concentration (25-200 microM) and uptake in each fraction. Linoleic acid uptake was equivalent in all fractions when expressed per mg protein, but when expressed in terms of the estimated minimal BBM, vesicle surface area uptake was greater in the upper- than in the lower-villus/crypt fractions. Thus, BBM vesicle uptake of both linoleic acid and glucose vary along the crypt-villus axis of the rabbit jejunum.

Dietary omega 3 fatty acids and cholesterol modify enterocyte microsomal membrane phospholipids, cholesterol content and phospholipid enzyme activities in diabetic rats.

Diabetes-associated changes in intestinal uptake of nutrients are modified by isocaloric variations in the type of dietary lipids, and are associated with alterations in the phospholipid and fatty acyl content of the intestinal brush border membrane. The present study was designed to test the hypothesis that diet- and diabetes-associated changes in enterocyte microsomal membrane phospholipids are due to variations in the activity of two phospholipid metabolizing enzymes, 1,2-diacylglycerol:CDPcholine cholinephosphotransferase (CPT) and phosphatidylethanolamine methyltransferase (PEMT). Adult female Wistar rats were fed one of four semisynthetic diets--beef tallow low in cholesterol (BT), beef tallow high in cholesterol (BTC), fish oil low in cholesterol (FO) or fish oil high in cholesterol. In half of the animals, diabetes mellitus was produced by injection of streptozotocin. Jejunal and ileal enterocyte microsomes (EMM) were isolated and analyzed for cholesterol and phospholipids, as well as for CPT and PEMT activities. In control animals, feeding FO reduced EMM total phospholipids including phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol. Feeding FO resulted in a greater than 95% reduction in the activity of CPT. Diabetes was associated with increased jejunal EMM total phospholipids including sphingomyelin (SM) and PE, without associated changes in CPT or PEMT. Dietary cholesterol supplementation did not affect EMM total cholesterol or phosphlipid composition in control rats fed BT or FO, but was associated with an increase in EMM cholesterol in diabetic rats fed BT or FO. A decrease in total phospholipids due to a decline in SM, PC and PE in diabetic rats fed FO was not associated with changes in the activities of CPT or PEMT in EMM.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of an alphaprodine-hydroxyzine combination as a sedative agent in the treatment of the pediatric dental patient.

This investigation proposed and evaluated a submucosally administered standard dose drug combination containing .6 mgm/kgm alphaprodine hydrochloride and .3 mgm/kgm hydroxyzine hydrochloride, in conjunction with nitrous oxide, oxygen, and lidocaine with epinephrine, in the sedation and treatment of uncooperative pediatric dental patients. Observations from this study include: The technique was successful in attaining a desirable, conscious sedation level that was characterized by rapid onset, maintenance throughout treatment, and rapid reversibility with naloxone and 100% oxygen. The protective airway reflexes remained intact during the procedures. Vital signs remained fairly constant throughout the treatment. Respiratory rate was slightly depressed during the sedation, but hemoglobin oxygen saturation levels remained constant. There were no incidences of respiratory depression, hypoxia, or apnea either during or after treatment, and no emergency medical or resuscitative efforts were necessary. Twenty-four hours after the appointment, few patients complained of the experience and more than half stated that they looked forward to returning. In view of these findings, the techniques and drug combination described in this investigation appear to provide a safe and effective means for the sedation and treatment of uncooperative pediatric dental patients. It should be emphasized, however, that the dentist using these methods should have a thorough knowledge of the agents involved, the ability to monitor patients and recognize possible adverse reactions, and the capacity to respond to any emergency situations should they arise. Consequently, only practitioners who have had extensive training and experience in all forms of anesthesia, especially pediatric anesthesia, should consider the use of these agents in their practices. It should be emphasized further that the agents and techniques should be used only for conscious sedation, the light level of sedation during which the patient retains the ability present before sedation to independently maintain an airway and respond appropriately to verbal command, and not for any deeper forms of anesthesia.

[Growth of the fetal thyroid gland in the 2nd half of pregnancy--biometric ultrasound studies]. / Wachstum der fetalen Schilddrüse in der II. Schwangerschaftshälfte-Sonographisch-biometrische Untersuchungen.

PURPOSE: Determination of normal values for fetal thyroid size in the 2nd half of pregnancy. METHOD: In 211 pregnant women (completed 20th to 41st week for pregnancy) without thyroid disease the fetal thyroid was measured and characterized by 5 parameters: right lobe transverse and p.a. diameter, left lobe transverse and p.a. diameter, right lobe and left lobe transverse including trachea. The level of measurement was a horizontal section of the neck with circular image of the trachea (centrally) and with the carotids as lateral limiting markers. RESULTS: The trend lines and regression coefficients of the parameters are shown in relation to week of pregnancy. The transverse diameter of both thyroid lobes including the trachea grows from 10 mm (20th week of pregnancy) to 20 mm (40th week of pregnancy). The transverse and p.a. diameters of either lobe double in the 2nd half of pregnancy. CONCLUSION: The normal values presented can help in early diagnosis of fetal thyroid hypertrophy.

On the use of tryptophan to detect slow orientational motions.

For our investigations on orientational motions of membrane proteins with the method of time-resolved fluorescence anisotropy decay (TRFA), we first wanted to test the use of tryptophan fluorescence in detecting slow motions on the time scale of tens of nanoseconds and to get more insight into the possible motions of membrane proteins by investigating a simple system. We performed TRFA measurements on a short α-helical 21-amino acid peptide in different environments (Vogel, H.,et al. Proc. Natl. Acad. Sci. USA 85, 5067-5071, 1988). In each case, we got three relexation time constants. The longest of these depends strongly on changes in the environment, whereas the two shorter times show only weak dependencies. So we conclude that the longest time belongs to the rotational diffusion of the entire peptide and the other to internal motions.

Feeding trans fatty acids to rats has no effect on the intestinal uptake of glucose, fatty acids or cholesterol.

Trans fatty acids are produced in the manufacture of margarine, and these hydrogenated fatty acids may have a deleterious effect on the reduction in fasting levels of serum cholesterol anticipated from the feeding of cis polyunsaturated fatty acids. We undertook this study in rats to test the effect of feeding trans fatty acids on the intestinal uptake of glucose, fatty acids and cholesterol. Adult female Wistar rats were fed for 2 weeks semisynthetic, isocaloric diets containing no oleic acid (18:1), cis 18:1 or trans 18:1. There was no difference between the three dietary groups in the animals' food consumption or body weight gain. Rats fed trans 18:1 had an approximately 20% decline in the total weight of the ileum as compared with controls fed no 18:1, and therefore there was also a decline in the percentage of the ileal tissue comprised of mucosa. When comparing rats fed trans 18:1 with those fed cis 18:1 or no 18:1, there was no difference in the uptake of varying concentrations of D-glucose when expressed as nmol.100 mg tissue-1.min-1 or nmol.100 mg mucosal-1.min-1 for jejunum or for ileum. Also, there was no difference in the value of the maximal transport rate (Vmax), Michaelis constant (Km), or the contribution of passive uptake of glucose assessed with L-glucose. There was no diet-associated change in the jejunal or ileal uptake of a medium-chain length fatty acid (lauric acid), a long-chain length saturated fatty acid (palmitic acid), a monounsaturated fatty acid (oleic acid), two polyunsaturated fatty acids (linoleic and linolenic acids), or cholesterol. Thus, we conclude that 2 weeks' feeding of trans fatty acid to rats has no influence on the jejunal or ileal uptake of glucose, fatty acids or cholesterol.

A long lifetime component in the tryptophan fluorescence of some proteins.

The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T1, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T1, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypeptide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.

Age of rat influences isolation of jejunal enterocytes from along the villus.

Aging is associated with alterations in the functions of the intestine. Also, the functions of the intestine vary along the length of the villus. A method is reported for the isolation of four fractions of enterocytes from along the villus of young (3 months, 300-350 g) and old (8 months, 700-750 g) male Wistar rats. Different qualitative patterns are seen when the activities of alkaline phosphatase (AP), invertase (INV), or [3H]methylthymidine incorporation are expressed on the basis of millimoles per gram protein, percentage of highest activity, percentage of total activity, cumulative activity for each fraction, or millimoles per gram protein versus cumulative percentage of total protein. When expressed on the basis of protein, AP is higher in upper (FI) versus lower (FIV) villus fractions, and in each fraction AP is higher in old versus young animals. INV varies little along the villus or between young and old rats; [3H]methylthymidine incorporation is highest in the crypt cells. The ratio mg protein/mg DNA varies along the villus and is twofold higher in young than in old rats. In contrast, when activities are expressed on the basis of DNA, the same qualitative patterns are seen along the villus, but INV and AP are both higher in young than in old rats. In summary (i) the qualitative and quantitative differences in the activities of AP and INV along the villus of old versus young rats vary when the data are expressed on the basis of DNA rather than protein; (ii) the ratio of protein to DNA varies along the villus in young and old animals, and for this reason it is appropriate to report enzyme activity on the basis of enterocyte DNA; and (iii) INV and AP, when expressed on the basis of DNA, are higher in young than in old animals. Thus, the isolation of rat jejunal enterocytes from along the villus is affected by the age of the animals.

Nutrient uptake into undifferentiated and differentiated HT-29 cells in culture.

HT-29 human colon carcinoma cells in culture have many characteristics of enterocytes, and these cells have been used by others to study intestinal drug and nutrient transport and metabolism. When grown in glucose-containing medium, HT-29 cells are largely undifferentiated (HT-29glu), but when grown in the absence of glucose but in the presence of galactose (HT-29gal), the population of cells is mostly differentiated. This study was undertaken with HT-29glu and HT-29gal cells to study the uptake of palmitic acid (16:0), linoleic acid (18:2), and cholesterol. The relationship between concentration and uptake of 16:0, 18:2, and cholesterol was linear in HT-29glu and HT-29gal cells, with the relative values of the slopes of this relationship being 18:2 > > 16:0 > > cholesterol. The rates of uptake of these lipids were at least three times higher in HT-29gal than in HT-29glu cells. In HT-29glu cells, the relative rates of uptake of the sugars at 32 mM were D-glucose = galactose > fructose > > alpha-methylglucose. Uptake of these sugars was much greater in HT-29gal than in HT-29glu cells. When 100 microM forskolin was added to the incubation medium for 7 days post-confluency, which stimulates the activity of adenylate cyclase and thereby increases the intracellular synthesis of cAMP, there was no effect on the uptake of the lipids or the sugars in either HT-29glu or HT-29gal cells. Thus, (i) differentiated HT-29gal cells transport larger amounts of lipids and sugars than do undifferentiated HT-29glu cells; (ii) forskolin has no effect on the uptake of lipids or sugars in these cells. This human cell culture system may be useful to study the in vitro transport of lipids, to establish the role of cell differentiation on these uptake processes, and to determine the potential role of selected intracellular signals.

The use of a long-lifetime component of tryptophan to detect slow orientational fluctuations of proteins.

The membrane protein porin and a synthetic polypeptide of 21 hydrophobic residues were inserted into detergent micelles or lipid membranes, and the fluorescence of their single tryptophan residue was measured in the time-resolved and polarized mode. In all cases, the tryptophan fluorescence exhibits a long-lifetime component of about 20 ns. This long-lifetime component was exploited to detect slow orientational motions in the range of tens of nanoseconds via the anisotropy decay. For this purpose, the analysis of the anisotropy has to be extended to account for different orientations of the dipoles of the short- and long-lifetime components. This is demonstrated for porin and the polypeptide solubilized in micelles, in which the longest relaxation time reflects the rotational diffusion of the micelle. When the polypeptide is inserted into lipid membranes, it forms a membrane-spanning alpha-helix, and the slowest relaxation process is interpreted as reflecting orientational fluctuations of the helix.