NPC1 plays a role in the trafficking of specific cargo to melanosomes.

Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.

Defining the altered glycoproteomic space of the early secretory pathway by class I mannosidase pharmacological inhibition.

N-glycosylation is a key process for various biological functions like protein folding, maturation and sorting for the conventional secretory compartment, cell-cell communication and immune response. This is usually accomplished by a complex system of mannosidases in which those from class I have an outstanding role, commonly involved in the early protein sorting associated to the Endoplasmic Reticulum (ER) in the N-glycan dependent quality control (ERQC) and ER-associated degradation (ERAD). Although these are vital processes in maintaining cellular homeostasis, large-scale analysis studies for this pool of molecules, further denoted as proteins from the early secretory pathway (ESP), were limited addressed. Here, using a custom workflow employing a combination of glycomics and deglycoproteomics analyses, using lectin affinity and selective Endoglycosidase H (Endo H) digestion, we scrutinize the steady-state oligomannosidic glycoprotein load and delineate ESP fraction in melanoma cells. All of these were assessed by applying our workflow for glycosite relative quantification of both the peptide chain and carbohydrate structure in cells with inhibited activity of class I mannosidases after kifunensine treatment. We found that most of the ESP are transient clients involved in cell communication via extracellular matrix, particularly integrin-mediated communication which adopt Man9 N-glycans in kifunensine-treated cells. Moreover, our results reveal that core-fucosylation is decreased subsequent inhibition of class I mannosidases and this could be explained by a general lower protein level of FUT8, the enzyme responsible for fucosylation. By comparing our data with results obtained following downregulation of a key mannosidase in misfolded protein degradation, we mapped both novel and previously suggested endogenous substrate candidates like PCDH2, HLA-B, LAMB2 or members of the integrin family of proteins such as ITGA1 and ITGA4, thus validating the findings obtained using our workflow regarding accumulation and characterization of ESP transitory members following mannosidase class I inhibition. This workflow and the associated dataset not only allowed us to investigate the oligomannosidic glycoprotein fraction but also to delineate differences mediated at glycosite-level upon kifunensine treatment and outline the potential associated cellular responses.

Profiling Optimal Conditions for Capturing EDEM Proteins Complexes in Melanoma Using Mass Spectrometry.

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.

Influence of light-dark cycle alteration on free radical level in rat cns.

The experiment analysed the evolution of several redox parameters caused by light-dark cycle alteration in different rat brain segments: cortex, brain stem, diencephalon. Continuous light (L:L) enhanced SOD, CAT, GPX and GSH levels to different extents in the various central nervous system regions investigated, depending on the entrainment period: 24 h, 48 h, 72 h, 7 days. Continuous darkness (D:D -24 h, 48 h, 72 h, 7 days) generally diminished SOD, CAT and GSH values, but increased GPX at 48 h and CAT at 72 h. MDA levels were higher in D:D, too. After 7 days L:L or D:D redox indices tended to reach the normal range again. Redox system spatio-temporal versatility and complementarity suggested the existence of a possible biochemical radical cell clock gear.

[The effect of the combination of vitamin E and C on the oxidative stress parameters in physical exercise]. / Cercetari privind efectul cuplului de vitamine E si C asupra unor parametri de stres oxidativ în exercitiul fizic.

The paper outlines the modification of some antioxidant enzymes and of reduced glutathione studied on physical training induced oxidative stress model. We also assessed vitamin E and C effect. Biochemical determinations were performed on heart homogenate and erythrocytes. Catalase and glutathione peroxidase activity diminished and superoxide dismutase activity increased to a different extent in both tissue samples, while coupled vitamin E and C protection to these tissues equally varied. The glutathione (GSH) pool decreased in erythrocytes and was moderately enhanced in the heart. Either in red blood cells or heart tissue GSH level constancy was maintained by simultaneous administration of vitamins through the experiment (training period). Malondialdehyde concentration revealed a slightly pro-oxidative behaviour of this couple of vitamins that explained the only partial recovery of enzymatic activity to normal values as well as a moderate lipid peroxidation process. Both phenomena were better expressed in erythrocytes.

Stimulation of the secretion of dehydroepiandrosterone by melatonin in mouse adrenals in vitro.

Adrenals of young adult male mice kept on a LD 12:12 lighting regimen for three weeks prior to study and harvested at four different circadian stages were incubated for 2 hours with 0.4 IU synthetic ACTH in 2 ml Krebs-Ringer buffer (KR), or with 50, 150, and 450 microM of melatonin in KR containing 0.4 IU ACTH. The addition of melatonin to ACTH leads to a dose dependent stimulation of production and/or secretion of DHEA into the incubation medium irrespective of the circadian stage of harvesting of the adrenals. This relationship is of interest in view of the simultaneous decrease of dehydroepiandrosterone and melatonin in the course of aging, and the effects of these compounds upon aging related changes.

Chronobiology in laboratory medicine.

A critical amount of information has accumulated over the last decades to allow the application of chronobiology to clinical and laboratory medicine. The tasks faced in laboratory medicine include the quantitative measurement of the multifrequency human time structure in health and disease. For this purpose, it is essential to choose an adequate sample size in order to obtain meaningful results and quantitative endpoints which can be interpreted by inferential statistical techniques. No statistical technique is applicable for all purposes and it is essential that the assumptions underlying each technique and its limitation are well known to the investigator. The multifrequency nature of the human time structure has to be kept in mind in order to avoid erroneous results. Time qualified reference ranges have to be established for high amplitude rhythms. Circadian and/or circannual rhythm alterations have been described as group phenomenon in subjects with epidemiologically determined risk states for common diseases, but will require much further studies for the application to individual subjects. Rhythm parameters are new endpoints in the evaluation of the human time structure in health. Alterations of these parameters may occur as cause or as consequence of disease. Recognition of rhythm abnormalities in disease are critical for a meaningful application of chronopharmacology. Time dependent changes in pharmacokinetics and pharmacodynamics have to be taken into account in the interpretation of drug level determinations. A considerable degree of individuality of timing has been documented in some frequencies. This individuality and the rhythm abnormalities found in disease require the study of reference or marker rhythms. If the complexity of the human time structure is clearly understood and its study pursued in a critical manner with quantitative endpoints, chronobiology opens a new dimension in laboratory and clinical medicine.

Chronobiology of pituitary-thyroid functions.

One hundred ninety four children, 11 +/- 1.5 years of age and 166 elderly men and women, 77 +/- 8 years of age were studied over one or (in the case of some of the elderly subjects) over several (up to 4) 24-hours spans. All subjects were diurnally active and rested at night and followed their regular three meal pattern. The subjects were studied in subgroups of 20-25 during all four seasons of the year. During each study, blood was collected at 4 hour intervals over one 24-hour span (6 samples). Circadian and circannual variations were found and described by cosinor analysis in the children as well as in the elderly subjects. The children with endemic goiter (134) as compared to those without endemic goiter (60) showed a slight circadian phase advance in plasma total and free T3, a lower circadian amplitude of total T4 concentrations and the absence of a detectable circadian rhythm in free T4. The children with goiter showed a phase delay in serum TBG. There was no difference between the children with and without goiter in the circadian MESOR of any thyroid parameter or of TSH. The children with endemic goiter in the region of Dimbovita, Romania, are in clinical and biochemically euthyroid condition with some slight poral abnormalities of thyroid function. Seasonal variations in children and elderly patients showed the highest values of TSH during summer and fall, while the highest values in the plasma concentrations of thyroid hormones were found during the cold season of the years. Thyroglobulin in the children showed a circadian rhythm but no seasonal variation.

Circadian, weekly, and seasonal variations in cardiac mortality, blood pressure, and catecholamine excretion.

Time of occurrence of cardiac death due to arrhythmia, heart failure, or acute myocardial infarction was recorded in 86 elderly subjects, belonging to a group in whom circadian and circannual rhythms in blood pressure and urinary catecholamine excretion had been studied previously. All patients were retired, with no work responsibilities, and lived--closely-supervised in a home for the aged--on a routine that provided little differences between weekdays and weekends. Cardiac mortality showed a circadian variation, with a peak in the early morning hours, coinciding with the circadian peak in systolic and diastolic blood pressures. A weekly (circaseptan) variation in cardiac mortality was found, with the greatest number of patients dying on Mondays and the least on Thursdays. There were seasonal differences in cardiac mortality, with a peak in July and a broader peak during the cold season (December to February). The former coincides with the circannual peak in diastolic blood pressure, but is unrelated to the seasonal variation in norepinephrine excretion. Circadian, circaseptan, and circannual variations in cardiac mortality appear to be the expression of time-dependent, transient risk states for catastrophic cardiac events, which may lend themselves to preventive treatment.

Circadian rhythm parameters of endocrine functions in elderly subjects during the seventh to the ninth decade of life.

The circadian rhythm of 17 endocrine parameters (ACTH, aldosterone, cortisol, C-peptide, DHEA-S, FSH, growth hormone, insulin, LH, 17-OH progesterone, prolactin, testosterone, total T3, total T4 and TSH and estradiol and progesterone in women only) were studied in 63 clinically apparently healthy men (124 profiles) and 86 women (154 profiles) during the 7th to 9th decade of life. The subjects lived under very uniform conditions in a home for the aged with their daily schedule standardized by institutional routine with rest at night on the average from 21:30 to 06:30 local time and 3 daily meals at 08:30, 13:00 and 18:30. Blood was drawn over a 24-h span at 4-h intervals. Circadian periodicity was ascertained and the rhythm parameters quantified by cosinor analysis. In clinically healthy elderly subjects, circadian periodicity persisted in most parameters studied well into the 9th decade of life. The timing of the circadian rhythm was comparable between subjects in their 7th decade and 9th decade of life with the exception of cortisol and DHEA-S, which showed a phase advance with advancing age. A decrease in circadian amplitude is limited during this part of the human life span to only a few of the functions investigated and with the exception of prolactin in the women, a decrease in amplitude did accompany a decrease in MESOR.

Circadian and seasonal variations in iodine excretion in children with and without endemic goiter.

The urinary iodine excretion was measured in 193 children 11 +/- 1.5 years of age living in the endemic goiter area of Dîmbovita, Romania. One hundred and thirty four of the children showed some degree of endemic goiter, 59 showed none. All children followed a diurnal activity pattern with rest during the night. They received their usual iodine supplement of 1 gm potassium iodide once a week during the school year (which included the time of all measurements made). Urine was collected in six 4-hour samples over a 24-hour span. The examinations were conducted during the months of March, June, September and December. Iodine was determined by an automated ceric ion arsenic acid method using a Technicon Autoanalyzer. Circadian and seasonal variations of urine volume and iodine excretion were statistically verified by the cosinor technique and the seasonal variations also by one way analysis of variance using the circadian means as input. A comparable circadian rhythm of iodine excretion was found in the children with and without endemic goiter, with an acrophase during the evening (20:16 with a 95% C.I., from 19:32 to 21:04). The circadian rhythm in iodine excretion has to be taken into account whenever an estimate of the 24-hour excretion is attempted from a sample covering less than the entire 24-hour span. There was a statistically significant seasonal variation of the 24-hour iodine excretion in the boys with and without endemic goiter and in the group as a whole. The 24-hour iodine excretion during March was 102 +/- 6 mcg, during June 81 +/- 4 mcg, during September 79 +/- 3 mcg and during December 102 +/- 7 mcg. The average 24-hour iodine excretion pooled over all seasons was 91 +/- 3 mcg/24 hrs in the children with and 91 +/- 5 mcg/24 hrs in the children without endemic goiter. During March and December the iodine excretion indicates an iodine intake not usually associated with a high prevalence of endemic goiter. However, during the months of June and September (and presumably even more during the months of July and August when during summer vacation no iodine supplementation was given in school) the 24-hour iodine excretion indicates some degree of iodine deficiency. The seasonal variation in urinary iodine excretion thus points to a time when increased iodine prophylaxis may be of value.

Chronobiology of serum iron concentration in subjects of different ages at different geographic locations.

The circadian rhythm in serum iron concentration was studied in 61 elderly men (74 +/- 6 years of age) and 93 women (78 +/- 8 years of age) in Bucharest, Romania, in 81 clinically healthy boys and 103 girls (11 +/- 1.5 years of age) in Tîrgoviste, Romania, in 4 elderly men and 19 women (71 +/- 5 years of age) and in 75 young-adult men (24 +/- 11 years of age) and 52 women (24 +/- 9 years of age) in St. Paul, Minnesota, USA. Six samples were obtained from each subject around a 24-hour span. The sampling sessions in the elderly subjects in Romania and in the children extended over all four seasons. A circadian rhythm statistically verified by Cosinor analysis was evident in all groups in both locations. A statistically significant sex difference with lower circadian mean (mesor) and a lower amplitude in the women was found in the Romanian elderly subjects. The children in Romania showed no sex difference in any circadian rhythm parameters. The young adult subjects in Minnesota showed a significantly higher mesor and a phase delay in the men as compared with the women. The elderly subjects of both sexes at both geographic locations had a lower circadian mesor than the young adults and the children. In the Romanian elderly subjects also the circadian amplitude was lower, which was not the case in the Minnesotans. While the acrophase in the elderly subjects and in the children in Romania was comparable (0928 and 0932 local time resp.), the young adults in Minnesota showed in comparison to the Romanians a phase delay (1132 local time) and the elderly in Minnesota showed a phase advance (0732 local time) in comparison to all other groups. The latter finding will have to be confirmed by more extensive studies. In the elderly subjects in Romania the circadian rhythm in serum iron concentration was in phase with the circadian rhythms in total serum bilirubin and alkaline phosphatase but showed significant phase differences from the circadian rhythms in serum albumin, urea nitrogen (BUN), gammaglutamyl transferase (Gamma-GT), serum globulins, glucose, insulin and total serum proteins. The elderly subjects in Romania showed a statistically significant circadian phase delay in summer as compared to fall but showed no seasonal variation of the mesor. The children showed a circadian phase advance in fall as compared to the other seasons and a seasonal variation of their mesor with higher values in spring and summer as compared with winter and fall.(ABSTRACT TRUNCATED AT 400 WORDS)

Circadian and circannual variations of thyroid function in children 11 +/- 1.5 years of age with and without endemic goiter.

A group of 194 children 11 +/- 1.5 years of age from Tirgoviste, Romania, an endemic goiter area, were studied over a 24-hr span (six blood samples at 4-hr intervals) during all four seasons. One hundred thirty-four of the children had some clinical evidence of endemic goiter, and 60 had none. Total and free T3 and T4, reverse T3, thyroglobulin, thyroxin-binding globulin (TBG; three seasons only), and TSH were studied. The circadian rhythms were analyzed by cosinor and the circannual variations by ANOVA. Children with and without endemic goiter showed circadian rhythms in all functions studied except free T4 for which no statistically significant rhythm was detected in the children with goiter. There were differences in the acrophase of total T3, free T3, and TBG, with phase advance in the children with goiter in total T3 and free T3 and a phase delay in TBG. Mesor and amplitude showed no differences except in total T4 for which the amplitude in the children with goiter was statistically significantly lower than in the children without goiter. Children with and without endemic goiter showed seasonal variations in total T4 and free T4 as well as total T3, free T3, and reverse T3, with the highest values in the fall; in thyroxin-binding globulin the highest values were in the winter; and in TSH the highest values were in the summer. There was no significant seasonal variation in thyroglobulin. There was no difference in the circannual variation between children with and without endemic goiter.

Circannual variations in blood pressure, urinary catecholamine excretion, plasma aldosterone, and serum sodium, potassium, calcium, and magnesium in children 11 +/- 1.5 years of age.

A total of 194 clinically healthy children 11 +/- 1.5 years of age were studied during different seasons. Systolic and diastolic blood pressure were determined by auscultatory endpoints, and blood and urine were collected at 4-hr intervals over a 24-hr span. Urinary norepinephrine, epinephrine, and dopamine were determined by HPLC, plasma aldosterone by RIA, serum sodium and potassium by ion-specific electrode, and calcium and magnesium by colorimetry. The circadian means showed statistically significant circannual variations in all variables except epinephrine. The highest circadian means of systolic and diastolic blood pressure and of urinary norepinephrine occurred during winter; the highest values of plasma aldosterone, serum potassium, and urinary dopamine were found in fall, those of serum calcium and magnesium during the summer, and that of serum sodium in spring. Circannual rhythms characterize functions related to blood pressure regulation in children. The circannual elevation of blood pressure (within the usual range) and of norepinephrine were both found to occur in winter. This time relation may have a functional significance, although a causal relationship is not proven by the temporal coincidence of two rhythms.

Chronobiology of catecholamine excretion in different age groups.

Urine was collected at 4-hr intervals over a 24-hr span in 87 boys and 106 girls 11 +/- 1.5 years of age and over one or several 24-hr spans in 62 elderly men and in 85 elderly women 77 +/- 8 years of age. Epinephrine, norepinephrine, and dopamine were determined by HPLC. The data were analyzed by cosinor and by one-, two-, and three-way ANOVA. Children and elderly subjects showed circadian rhythms of urine volume and of the excretion of norepinephrine, epinephrine, and dopamine. While the urine volume was higher in the elderly subjects than in the children, norepinephrine, epinephrine, and dopamine excretion in the girls and epinephrine in the boys showed a statistically significantly higher mesor than in the elderly subjects of the same sex. There was a sex difference, with lower values in all variables in the girls and women compared to their male counterparts; the circadian amplitudes of norepinephrine, epinephrine, and dopamine in the girls and of epinephrine in the boys were higher than the circadian amplitudes in the elderly subjects. The circadian timing in urinary excretion between the elderly subjects and the children was different, with a consistent phase delay; the acrophase of the circadian rhythm in the elderly subjects moved in the night hours. In contrast, there was no age difference in the acrophase of norepinephrine and epinephrine excretion or in dopamine in the females. In the males, the circadian rhythm in dopamine excretion in the elderly subjects did not quite reach statistical significance at the P less than 0.05 level. Circannual variations with high values in winter and low values in spring and summer were found in norepinephrine excretion in boys, girls, and elderly women, but not in elderly men. In neither age group was there a statistically significant seasonal variation in epinephrine. Only in girls was a statistically significant circannual rhythm in dopamine excretion found, with highest dopamine values in the fall and lowest values in winter and spring.

Circannual rhythms of laboratory measurements in serum of elderly subjects.

A total of 160 elderly subjects 77 +/- 8 years of age were studied in 201 24-hr profiles consisting of six blood samples collected at 4-hr intervals. The sampling sessions were spread over all four seasons. The circadian means were analyzed (one-way ANOVA) for the presence of circannual variations. Statistically significant circannual variations were found in the serum concentrations of albumin, bilirubin, calcium, chloride, CPK, globulin (calculated), glucose, LDH, potassium, sodium, triglycerides, uric acid, and total protein. The data presented indicate that many chemical constituents commonly measured in human serum and urine show seasonal variations in elderly subjects, some of which are large enough to present potential diagnostic problems. Others may not pose diagnostic problems in today's practice of laboratory medicine, but indicate seasonal changes in metabolic functions or, if endogenous in nature, circannual rhythms that may be of physiologic and pathobiologic importance. There is a need to quantify certain of these rhythms as a predictable portion of variability in laboratory values and presumably as an indicator of human health.

Endocrine factors of blood pressure regulation in different age groups.

Systolic and diastolic blood pressures were measured and blood and urine were collected at 4-hr intervals over a 24-hr span in 194 diurnally active children 11 +/- 1.5 years of age and in 278 elderly subjects 77 +/- 8 years of age. Plasma aldosterone and cortisol were determined by radioimmunoassay, serum calcium and magnesium on a Dupont ACA, and urinary epinephrine and norepinephrine by high-pressure liquid chromatography. In the children, there was a slight but statistically significant positive correlation between the circadian means in systolic blood pressure and norepinephrine excretion and serum calcium, and between diastolic blood pressure and norepinephrine excretion and serum calcium and magnesium. In the elderly subjects, there was a positive correlation between the circadian mean in diastolic blood pressure and aldosterone. In contrast to the findings in the children, however, the elderly subjects showed a negative correlation between the circadian means in norepinephrine excretion and in systolic and diastolic blood pressures. These investigations indicate differences in the regulation of the blood pressure within the "usual range" between children and elderly subjects. This has to be kept in mind in the study of essential hypertension, a syndrome that may be caused by different mechanisms in different age groups.