Evidence for Structural Dissimilarity in the Neurotransmitter Binding Region of Purified Acetylcholine Receptors from Human Muscle and Torpedo Electric Organ

Acetylcholine receptor (AChR) purified from human skeletal muscle affinity-alkylated with bromoacetyl[methyl-3H]choline bromide ([3H]BAC) in mildly reducing conditions to yield a specifically radiolabeled polypeptide, Mr 44,000, the alpha-subunit. The binding of [125I]alpha-bungarotoxin to AChR was completely inhibited by affinity-alkylation, indicating that the human AChR's binding site for alpha-bungarotoxin is closely associated with the alpha-subunit's acetylcholine binding site. Structures in the vicinity of the alpha-bungarotoxin binding sites of AChRs from human muscle and Torpedo electric organ were compared by varying the conditions of alkylation. Under optimal conditions of reduction and alkylation, both human and Torpedo AChR incorporated BAC in equivalence to the number of alpha-bungarotoxin binding sites. However, with limited conditions of reduction but sufficient BAC to alkylate 100% of the alpha-bungarotoxin binding sites of human AChR, only 71% of the Torpedo AChR's binding sites were alkylated. In optimal conditions of reduction but with the minimal concentration of BAC that permitted 100% alkylation of the human AChR's alpha-bungarotoxin sites, only 74% of the Torpedo AChR's binding sites were alkylated. These data suggest that the neurotransmitter binding region of human muscle AChR is structurally dissimilar from that of Torpedo electric organ, having a higher binding affinity for BAC and an adjacent disulfide bond that is more readily accessible to reducing agents.     

Microphytobenthic biomass assessment by pigment analysis: comparison of spectrophotometry and High Performance Liquid Chromatography methods

Samples of microphytobenthos from the Tagus estuary were analysed for photosynthetic pigments by spectrophotometry and High Performance Liquid Chromatography (HPLC). Chlorophyll a values obtained with HPLC and spectrophotometry methods presented a highly significant positive correlation for both spectrophotometric methods used (with and without the correction for pheopigments), but this relationship depended on the type of sediment. We concluded that spectrophotometric methods give reliable Chl-a values, being suited for routine analysis, when a vast number of replicates is needed. However, for the correct estimation of pheopigments, HPLC analysis is indispensable. In the literature, Chl-a estimations are expressed per content (μg g⁻¹) or concentration (mg m⁻²). We discuss the influence of sediment type on the results depending on the type of unit used, and propose a simple conversion factor based on sediment water content.     

Novel redistribution of myosin-containing filaments in cultured keratinocytes identified by a human monoclonal autoantibody

Summary A time-dependent redistribution of microfilaments was observed in cultured human keratinocytes using a human monoclonal autoantibody specific for myosin. Immunofluorescent staining revealed that 5 days after plating keratinocytes in either 0.1 mM or 2.0 mM Ca⁺⁺, myosin was distributed uniformly throughout the cytoplasm. At day 6, parallel arrays of myosin-containing microfilaments were prominent in the cell peripheries. At day 7 the microfilaments formed circumferential rings. The distribution of the microfilaments was disrupted by cytochalasin but not by colchicine, indicating that this novel distribution of myosin was not dependent on colchicine-sensitive vimentin intermediate filaments. The time-dependent redistribution of myosin was not influenced by cell population density, cell shape or cell cycle phase, except for mitotic cells in which myosin was distributed diffusely through the cytoplasm. If, as suggested by Kolega (9), microfilaments align parallel to the direction of applied tension, the redistribution of myosin-containing microfilaments in cultured keratinocytes may reflect the increased tension between cells resulting from increasing strength of cell-cell junctions over time. In sectioned human skin, myosin was localized in the peripheral cytoplasm of stratified epidermal cells. Tensions arising from the numerous desmosomal junctions between cellsin vivo could account for this distribution of myosin. Supported by grant NS-23537 (V. A. L.) from the National Institutes of Health, Bethesda, MD, and by the Mayo Foundation. C. L. W. is recipient of the Kermit E. Osserman and Blanche McClure Fellowship, 1987, National Myasthenia Gravis Foundation.     

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

Spatio-temporal distribution of the microphytobenthic biomass in intertidal flats of Tagus Estuary (Portugal)

A study on spatio-temporal distribution of microphytobhethos in intertidal zones of Tagus Estuary was carried out from 1990 to 1992. Near Lisbon, Portugal, Tagus Estuary is a shallow mesotidal estuary, covering an area of 320 km². The intertidal area ranges from 20 to 40% off the total area and it is constituted mainly by mudflats. Intertidal flats are richly populated by microalgae, diatoms being the most important and ubiquitos group.Spatial variation of microphytobethos was studied in spring 1990, 21 different sites were sampled. Microphytobenthos biomass was evaluated as chlorophyll a content of the surface centimeter, ranging from 10 to 240 mg m^–2. A Principal Component Analysis showed that 62% of the total variability found in intertidal flats of Tagus estuary could be attributed to two major factors: sediment type and tidal height. A hierarchical grouping defined 3 major groups of similar stations, each one representing a different strata of the ecosystem.One station from each group was chosen for the study of the temporal variation. A sampling, rogram took place from April 1991 to April 1992, with fortnightly sampling, the Chl a ranged from 20–300 mg m^–2. No clear seasonal variation was found, and our results indicated that tidal height of sampledsite played an essential role in temporal biomass evolution, thus upper littoral sites were influenced by climatic parameters, whereas in lower sites action of tides mainly controlled microphytic biomass.

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Résumé Une étude sur l'hétérogénéité spatio-temporelle du microphytobenthos dans les sédiments intertidaux de l'Estuaire du Tage a été accompli de 1990 á 1992.L'Estuaire du Tage, prés de Lisbonne (Portugal) est un estuaire peu profond, mesotidal, avec une aire total de 320 km². L'aire intertidale est comprise entre 20 et 40% du total, et constituéé surtout par des vasiéres. Ces slikkes sont peuplées par une communauté assez riche de microalgues, ou les diatomées sont les plus abundantes.La variation spatialle du microphytobenthos était évalué au Printemps 1990, ou 21 différentes stations étaient échantillonnées. La biomasse était évalué par la concentration enchlorophylle a du premier centimétre de sédiment, qui a varié de 10 á 240 mg Chl a m^–2. Une Analyse en Composants Principales a montré que 62% de la variabilité de la biomasse était lié á deux facteurs: le sédiment et l'hauteur vis-á-vis la marée. Une classification hiérarchique des stations par similitude a établi 3 groupes principaux, représantantles différents strates de écecosytéme.Une station de chaque groupement a été choisie pour l'étude de la variation temporelle, qui s'est deroulé d'avril 1991 á avril 1992, avec des prélévements deux fois par mois. Les valeurs de Chl a obtenus vont de 20 á 300 mg m^–2. Les variations saisonniéres observées ne sont pas claires: nos résultats indiquent que l'hauteur de la station (m) joue un rôle essentiel dans l'évolution temporel de la biomasse, c'est á dire, la biomasse microalgal des sites du supra-littoral est influencié par les paramétres climatiques, tandis que dans l'infra-littoral c'est l'action des marées le facteur principal.

     

Glycosylated equine prolactin and its carbohydrate moiety

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities,G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about l/30th the mitogenic activity of bovine PRL; G-ePRL was approximately l/10th as active as ePRL. Glycosylation of G-ePRL at Asn³¹ was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29–37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man₃, GlcNAc₂, GalNAc₁, Fuc_0.6, Gal_0.2, NeuAc_0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn³¹ carbohydrate unit is a fucosylated complex mono- and/or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.     

Short-term effects of winter warming and acidification on phytoplankton growth and mortality: more losers than winners in a temperate coastal lagoon

Hydrobiologia - Changes in temperature and CO2 are typically associated with climate change, but they also act on shorter time scales, leading to alterations in phytoplankton physiology and...     

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods.

Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins.

Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 μmol/g, in the DM CAT group 1187 vs 382 μmol/g and in the non-DM CAT group 967 vs 252 μmol/g; p < 0.05 for all).

In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Parathyroidectomy Improves Survival In Patients with Severe Hyperparathyroidism: A Comparative Study

Background and objectives

Secondary hyperparathyroidism (SHPT) in CKD is associated with an increased risk for mortality, but definitive data showing that parathormone control decreases mortality is still lacking. This study aimed to compare the mortality of patients with severe SHPT submitted to parathyroidectomy(PTX) with those who did not have access to surgery.

Methods

This is a retrospective study in a cohort of 251 CKD patients with severe SHPT who were referred to a CKD-MBD Center for PTX from 2005 until 2012.

Results

Most of our patients had indication of PTX, but only 49% of them had access to this surgical procedure. After a mean follow-up of 23 months, 72 patients had died. Non-survivors were older; more often had diabetes, lower serum 25 vitamin D and mostly had not been submitted to surgery. The relative risk of death was lower in the PTX patients (0.428; 95% CI, 0.28 to 0.67; p<0.0001). After adjustments, mortality risk was dependent on age (1.04; 95% CI, 1.01 to 1.07; p = 0.002), 25 vitamin D (0.43; 95% CI, 0.24 to 0.81; p = 0.006) and no access to PTX (4.13; 95% CI, 2.16 to 7.88; p<0.0001). Results remained the same in a second model using the PTX date as the study start date for the PTX group.

Conclusions

Our data confirms the benefit of PTX on mortality in patients with severe SHPT. The high mortality encountered in our population is significant and urges the need to better treat these patients.     

BaSIQS - basic scale on insomnia complaints and quality of sleep: reliability,norms, validity,and accuracy studies,based on clinical and community samples

This research focused on the Basic Scale on Insomnia Symptoms and Quality of Sleep (BaSIQS), formerly validated in undergraduates using the Pittsburgh Sleep Quality Index (PSQI), and aimed to expand internal consistency analysis, examine thoroughly its validity, and determine its clinical accuracy. Considering objective and subjective measures, recruiting non-clinical and clinical samples, this research implemented a comprehensive approach to examine convergent and discriminant validity, confirmatory factor analyses, and the BaSIQS sensitivity and specificity. The BaSIQS was filled out along with the Insomnia Severity Index (ISI), questions on sleep-wake schedules, Composite Scale of Morningness (CSM) and Brief Symptom Inventory-18 (BSI-18) by 1198 adults, 18–64 years old, plus another 30 who wore actimeters, recruited in community settings. A clinical group of 30 chronic insomnia disorder patients also participated. Cronbach alpha coefficient was 0.80. A two-factor structure was confirmed. The association between BaSIQS and ISI was large, whereas actigraphy correlations were medium or small. Medium to non-significant correlations were found concerning conceptually different self-report measures. Comparing the clinic and control groups, the former showed poorer sleep, with a large effect size. Receiver operating characteristic analysis revealed an area under curve = 0.9, and an optimal cut-off score >15. In conclusion, results on reliability, validity, and accuracy provide support to the utility of the BaSIQS both in community and clinical settings, for research and practical purposes.