Simultaneous analysis of lysine, Nepsilon-carboxymethyllysine and lysinoalanine from proteins

Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.     

A cytoplasm to vacuole targeting pathway in P. pastoris

The cytoplasm-to-vacuole targeting (Cvt) pathway of Saccharomyces cerevisiae delivers aminopeptidase I (Ape1) from the cytosol to the vacuole, bypassing the normal secretory route. The Cvt pathway, although well-studied, was known only in S. cerevisiae. We demonstrate its existence in the methylotrophic yeast, Pichia pastoris, where it also delivers P. pastoris Ape1 (PpApe1) to the vacuole. Most proteins known to be required for the Cvt pathway in S. cerevisiae were, to the extent we found orthologs, also required in P. pastoris. The P. pastoris Cvt pathway differs, however, from that in S. cerevisiae, in that new proteins, such as PpAtg28 and PpAtg26, are involved. The discovery of a Cvt pathway in P. pastoris makes it an excellent model system for the dissection of autophagy-related pathways in a single organism and for the discovery of new Cvt pathway components.     

Diez recomendaciones para mejorar la enseñanza de la Geriatría en el pregrado

IntroductionPopulation ageing and scientific progress have made specific training in Geriatrics essential for any medical students. There are different international recommendations on the teaching of Geriatric Medicine at undergraduate level, but they are still far from being offered systematically. The aim of this work was to develop a National list of recommendations in order to improve training in Geriatric Medicine at the undergraduate level.Material and methodsA list of 10 recommendations was produced by reflecting on the Geriatric training situation in Spain in 2016/2017 and international recommendations. Consensus was reached among the members of the Spanish Society of Geriatrics and Gerontology (SEGG) working group on «Geriatric training at undergraduate level» through 2 meetings and online discussions.ResultsA list of 10 of recommendations on Geriatric training in Spanish Medical Schools is proposed, including areas such as, the status of the discipline in the curriculum, length, contents, practical training, lecturers, teaching methods, and innovation.ConclusionsThese recommendations are intended to improve Geriatrics training. They can be used by different Medical Schools either to adapt their programs or to start training in Geriatric Medicine.     

Assessing internet survey data collection methods with ethnic nurse shift workers

An increasing number of ethnic minorities are expected to enter the United States workforce based on projected demographic changes. This includes American Indian/Alaskan Native (AI/AN) nurses. Sociocultural influences on sleep disturbances, sleepiness, and other aspects related to shift-work tolerance are of unrecognized importance. More minority nurses are needed to provide culturally congruent care; however, AI/AN nurses represent less than 1% of nurses located throughout the American workforce. This article aims to verify the feasibility of Internet data collection (Web-based survey) methods and instrument stability as the first part of a two-phase study comparing individual differences and shift-work-related sleep disturbances between AI/AN and White non-Hispanic (WNH) nurses. In the first phase, an Internet survey was used to reach a cross-section of AI/AN and WNH nurses. The on-line survey was composed of accepted shift-work-related instruments. Items estimating sleep disturbances, sociocultural choices, time awareness, polychronicity, morningness/eveningness, ethnic identity, and demographic questions were asked. The survey was linked to a series of Web pages describing the study purpose, inclusion and exclusion criteria, consent form, Web survey, and the second phase of the study in which subjects were invited to participate in actigraphy measurements. The survey was pilot-tested for error codes, item confusion, length, and completion time. Forced-answer questions were added asking ethnicity, age group, license type, state where licensed, and legal name on nursing license before accessing the survey. Data were saved periodically, cued by the word “continue.” The database was located on a secure server and password protected. Nurses were recruited using published articles and printed advertisements, hospital e-mail systems, national nursing organization Web sites (minoritynurse.com; NANAINA.org), nursing Web site discussion groups, snow-balling, and word of mouth. The site was accessed 656 times with the Internet survey being completed by 138 WNH and 56 AI/AN nurses meeting the inclusion criteria. Except for the polychronicity measure (PAI3), instruments measuring time awareness, chronotype, and situational sleepiness achieved acceptable reliability coefficients with Internet data collection. Using pull-down menus would improve questions asking specific times. Internet data collection with different ethnic groups is possible; however, accessing the target population may be difficult. Despite extensive recruitment efforts, few AI/AN nurses participated. Computer literacy and failing to relate to the study's purpose may have limited the interest of the AI/AN nurses. It is possible to recruit nurse shift workers and collect individual difference and sleep disturbance data through the Internet; however, the researcher must remain vigilant throughout the process.     

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.     

The coelomycetous genera <Emphasis Type="Italic">Chaetomella</Emphasis> and <Emphasis Type="Italic">Pilidium</Emphasis> represent a newly discovered lineage of inoperculate discomycetes

The coelomycetous genera Chaetomella and Pilidium were determined to be closely related to each other, yet are recognized as distinct genera based on both morphological observations and rDNA sequence analyses. Analyses of the SSU and LSU of the nuclear ribosomal RNA genes suggest that Chaetomella and Pilidium along with Sphaerographium tenuirostrum and Synchaetomella lunatospora constitute a distinct lineage within the ascomycetes that is allied with the Leotiomycetes. Chaetomella and Pilidium both produce black pycnidia generally opening by a raphe, acropleurogenous conidiogenous cells, and non-septate, hyaline, usually fusiform to falcate, rarely ellipsoid, conidia. Pycnidia of Pilidium are smooth while those of Chaetomella have setae of various types. A Hainesia sporodochial synanamorph was observed in cultures of C. oblonga and C. raphigera similar to H. lythri, the synanamorph of P. concavum. Specimens of C. oblonga, type species of Chaetomella, C. acutiseta, C. circinoseta, C. raphigera, Pilidium acerinum, type species of Pilidium, and P. concavum were examined. Diagnoses and illustrations are provided for these species along with a key to the accepted species in both genera. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

Antibody to beta-amyloid protein increases acetylcholine in the hippocampus of 12 month SAMP8 male mice

Amyloid beta protein (Abeta) is the primary constituent of plaque seen in Alzheimer's disease. Abeta is proposed to play an etiological role in Alzheimer's disease and to be a cause of the decrease in the level of acetylcholine in the hippocampus. The SAMP8 strain of mouse develops age-related increases in Abeta and deficits in learning and memory by 12 months of age. We examined in 12 month old SAMP8 mice the effects of giving antibody to Abeta by septal or intracerebroventricular (ICV) injection on acetylcholine levels in the hippocampus. Antibody to Abeta increased acetylcholine in the hippocampus over 100% after ICV injection and over 200% after septal injection. Injection of rabbit serum, antibody directed towards mouse IgG, or a blocking antibody directed towards human interleukin-1beta were without effect. These results suggest that antagonism of Abeta increases acetylcholine concentrations in the hippocampus, an area important for learning and memory.     

Microrheology of human lung epithelial cells measured by atomic force microscopy

Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G(*)(omega)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1-100 Hz) and at different loading forces (0.1-0.9 nN) with atomic force microscopy. G(*)(omega) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G'(omega) increased with frequency following a power law with exponent approximately 0.2. The loss modulus G"(omega) was approximately 2/3 lower and increased similarly to G'(omega) up to approximately 10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G'(omega) and G"(omega). G(*)(omega) conformed to the power-law model with a structural damping coefficient of approximately 0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.