Hypochromicity in red blood cells: an experimental and theoretical investigation.

Multiwavelength UV-visible transmission spectrophotometry is a useful tool for the examination of micron-size particle suspensions in the context of particle size and chemical composition. This paper reports the reliability of this method to characterize the spectra of purified red blood cells both in their physiological state and with modified hemoglobin content. Previous studies have suggested the contribution of hypochromism on the particle spectra caused by the close electronic interaction of the encapsulated chromophores. Our research shows, however, that this perceived hypochromism can be accounted for by considering two important issues: the acceptance angle of the instrument and the combined scattering and absorption effect of light on the particles. In order to establish these ideas, spectral analysis was performed on purified and modified red cells where the latter was accomplished with a modified hypotonic shock protocol that altered the hemoglobin concentration within the cells. Moreover, the Mie theory was used to successfully simulate the spectral features and trends of the red cells. With this combination of experimental and theoretical exploration, definition of hypochromism has been extended to two subcategories.

Glucosamine-induced increase in Akt phosphorylation corresponds to increased endoplasmic reticulum stress in astroglial cells.

Increased glucose flux through the hexosamine biosynthetic pathway (HBP) is known to affect the activity of a number of signal transduction pathways and lead to insulin resistance. Although widely studied in insulin responsive tissues, the effect of increased HBP activity on largely insulin unresponsive tissues, such as the brain, remains relatively unknown. Herein, we investigate the effects of increased HBP flux on Akt activation in a human astroglial cells line using glucosamine, a compound commonly used to mimic hyperglycemic conditions by increasing HBP flux. Cellular treatment with 8 mM glucosamine resulted in a 96.8% +/- 24.6 increase in Akt phosphorylation after 5 h of treatment that remained elevated throughout the 9-h time course. Glucosamine treatment also resulted in modest increases in global levels of the O-GlcNAc protein modification. Increasing O-GlcNAc levels using the O-GlcNAcase inhibitor streptozotocin (STZ) also increased Akt phosphorylation by 96.8% +/- 11.0 after only 3 h although for a shorter duration than glucosamine; however, the more potent O-GlcNAcase inhibitors O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-d]-Delta2'-thiazoline (NAGBT) failed to mimic the increases in phospho-Akt indicating that the Akt phosphorylation is not a result of increased O-GlcNAc protein modification. Further analysis indicated that this increased phosphorylation was also not due to increased osmotic stress and was not attenuated by N-acetylcysteine eliminating the potential role of oxidative stress in the observed phospho-Akt increases. Glucosamine treatment, but not STZ treatment, did correlate with a large increase in the expression of the endoplasmic reticulum (ER) stress marker GRP 78. Altogether, these results indicate that increased HBP flux in human astroglial cells results in a rapid, short-term phosphorylation of Akt that is likely a result of increased ER stress. The mechanism by which STZ increases Akt phosphorylation, however, remains unknown.

Selective decrease of membrane-associated PKC-alpha and PKC-epsilon in response to elevated intracellular O-GlcNAc levels in transformed human glial cells.

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the beta-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 muM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-betaII that was maximal after 3 h (73+/-8%) and also produced a 48+/-15% decrease in membrane-associated PKC-epsilon after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-epsilon in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87+/-6% with STZ and 73+/-7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-alpha membrane levels by 9 h posttreatment (78+/-10% with STZ and 66+/-8% with PUGNAc) while neither compound produced any changes in PKC-betaII translocation. In addition, none of the three compounds affected membrane levels of PKC-iota. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.

UV-visible spectrophotometric approach to blood typing II: phenotyping of subtype A2 and weak D and whole blood analysis.

BACKGROUND: A recently introduced quantitative blood typing approach uses antibody-induced changes in the UV-visible spectra of blood. Changes in the blood spectra's slope, caused by RBC agglutination, are translated into a numerical agglutination index (AI). Comparing the AI value against an established threshold yields a "yes and/or no" output from which to determine the phenotype. The efficacy and flexibility of this approach with whole blood use and the ability to analyze weak D, A2, and A2B were examined. STUDY DESIGN AND METHODS: Two hundred randomly selected blood bank donor samples were coded and forward typed directly from whole blood by using the spectrophotometric analysis. Reverse grouping on plasma from each sample was carried out with a new modified procedure by using higher ratios of plasma to RBCs. Results were compared to typing by an FDA-cleared automated typing system. Twenty-seven weak D samples, 15 A2 and 12 A2B, were similarly analyzed from whole blood. PEG improved detection of weak D, A2 and A2B subtypes. RESULTS: All two hundred coded samples were accurately typed, yielding identical results to the blood bank analysis in both forward and reverse grouping. All the weak D samples and A2 and A2B samples were clearly identified, having AIs above the type threshold indicator value. CONCLUSION: Spectrophotometric blood typing successfully phenotyped ABO and D in 200 whole blood samples. Reverse grouping of plasma was equally successful. The same method can identify weak D and A2 and A2B subtypes.

Position of the American Dietetic Association: ethical and legal issues in nutrition, hydration, and feeding.

It is the position of the American Dietetic Association that the development of clinical and ethical criteria for the nutrition and hydration of persons through the life span should be established by members of the health care team. Registered dietitians should work collaboratively to make nutrition, hydration, and feeding recommendations in individual cases. Registered dietitians have an active role in determining the nutrition and hydration requirements for individuals throughout the life span. When patients choose to forgo artificial nutrition and hydration, or when patients lack decision-making capacity, and others must decide whether or not to provide artificial nutrition and hydration, the registered dietitian has an active and responsible professional role in the ethical deliberation around that decision. There is strong clinical, ethical, and legal support both for and against the administration of food and water when issues arise regarding what is or is not wanted by the patient and what is or is not warranted by empirical clinical evidence. When a conflict arises, the decision to administer or withhold nutrition and hydration requires ethical deliberation. The registered dietitian's understanding of nutrition and hydration within the context of nutritional requirements and cultural, social, psychological, and spiritual needs provides an essential basis for ethical deliberation on issues of nutrition and hydration.