Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine's medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.

Development of the ProPal-COPD tool to identify patients with COPD for proactive palliative care.

BACKGROUND: Our objective was to develop a tool to identify patients with COPD for proactive palliative care. Since palliative care needs increase during the disease course of COPD, the prediction of mortality within 1 year, measured during hospitalizations for acute exacerbation COPD (AECOPD), was used as a proxy for the need of proactive palliative care. PATIENTS AND METHODS: Patients were recruited from three general hospitals in the Netherlands in 2014. Data of 11 potential predictors, a priori selected based on literature, were collected during hospitalization for AECOPD. After 1 year, the medical files were explored for the date of death. An optimal prediction model was assessed by Lasso logistic regression, with 20-fold cross-validation for optimal shrinkage. Missing data were handled using complete case analysis. RESULTS: Of 174 patients, 155 patients were included; of those 30 (19.4%) died within 1 year. The optimal prediction model was internally validated and had good discriminating power (AUC =0.82, 95% CI 0.81-0.82). This model relied on the following seven predictors: the surprise question, Medical Research Council dyspnea questionnaire (MRC dyspnea), Clinical COPD Questionnaire (CCQ), FEV1% of predicted value, body mass index, previous hospitalizations for AECOPD and specific comorbidities. To ensure minimal miss out of patients in need of proactive palliative care, we proposed a cutoff in the model that prioritized sensitivity over specificity (0.90 over 0.73, respectively). Our model (ProPal-COPD tool) was a stronger predictor of mortality within 1 year than the CODEX (comorbidity, age, obstruction, dyspnea, and previous severe exacerbations) index. CONCLUSION: The ProPal-COPD tool is a promising multivariable prediction tool to identify patients with COPD for proactive palliative care.

Changes in the glycoprotein composition of plasma membrane during the differentiation of friend erythroleukemia cells.

Friend erythroleukemia cells display transient and permanent changes in the composition of their plasma membrane-bound glycoproteins during dimethyl sulfoxide-induced differentiation. The transient changes, as revealed by metabolic labeling with [14C]glucosamine, are most conspicuous around the time during which most cells become committed to terminal differentiation. Permanent changes are revealed by reductive tritiation after oxidation with NaIO4 or galactose oxidase. In differentiated cells one glycoprotein fraction (Mr 150 000) could not be labeled by any of these methods, although it does contain neuraminic acid. We found no evidence in support of the hypothesis that the anomalous behavior of this fraction is caused by an increased degree of O-acetylated neuraminic acid in the plasma membrane of differentiated cells.

Inhibitors of protein glycosylation inhibit the differentiation of Friend erythroleukemia cells.

Tunicamycin, 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-glucose inhibit dimethyl sulfoxide-induced differentiation of Friend cells. This inhibition, characterized by inhibition of hemoglobin synthesis, is accompanied by a specific inhibition of protein glycosylation. The results of cloning experiments indicate that this inhibition specifically affects cells in the period preceding their commitment. These results suggest that glycoprotein synthesis is a requirement for Friend erythroleukemia cells in order to initiate the expression of the terminal differentiation program.

Storage-related changes in erythrocyte band 3: not a case for the Diego blood group antigens.

Removal of erythrocytes from the circulation is mediated by the immune system. Changes in structure and function of band 3, a major membrane protein of the erythrocyte, trigger the binding of antibodies to a band 3-derived neoantigen, senescent cell antigen, on erythrocytes aged in vivo. This mechanism probably is also involved in determining the survival of erythrocytes after transfusion. Band 3 is the carrier of the Diego blood group system, and subtle changes in the three-dimensional conformation of the same extracellular loops of band 3 determine Diego blood group activity as well as senescent cell antigen activity. Therefore we used the Diego blood group system to probe these changes with a combination of serological and immunochemical methods. Our data indicate that changes in band 3 structure during storage under blood bank conditions, as shown by immunoblot analysis, are not detectable as changes in expression of Diego antigens in intact cells. This makes it unlikely that immunological removal of erythrocytes after transfusion is mediated by reactions involving the Diego blood group system.

Naturally occurring human "antigalactosyl" IgG antibodies are heterophile antibodies recognizing blood-group-related substances.

IgG autoantibodies in human serum bind selectively to a new antigen that appears on senescent erythrocytes, thereby initiating their removal from the circulation. We tested the hypothesis that these IgG molecules recognize terminal galactose residues, thought by some investigators to become exposed as cells age. Results revealed that human antibodies with an "antigalactosyl" specificity are heterophile antibodies directed against rabbit and not human red cells. This was demonstrated using hemagglutination assays and immunoblotting. Immunoblots performed with affinity purified antigalactosyl antibodies revealed binding of the antibodies to rabbit, but not human, erythrocyte membrane proteins. They have a broad range of specificities including anti-B, anti-I, and possibly anti-P1 and Pk. These heterophile antibodies are not involved in the physiological removal of senescent human RBC by macrophages as indicated by the data demonstrating that incubation of senescent red cells aged in situ with galactose prior to incubation with macrophages does not alter their phagocytosis. Senescent cell IgG does not have an antigalactosyl specificity because IgG eluted from senescent cells aged in situ does not bind to rabbit red cells that have exposed alpha-galactosyl moieties.

Erythrocyte membrane characteristics indicate abnormal cellular aging in patients with Alzheimer's disease.

Erythrocytes from patients with Alzheimer's disease show signs of disturbance of the normal cellular aging process. Immunoblotting of erythrocyte membrane proteins from Alzheimer patients reveals increased breakdown of the anion transport protein band 3 in a majority of the cells, similar to what is observed in only a very small cell population during normal aging. These structural changes are accompanied by changes in anion transport characteristics, but the latter partially deviate from those observed during normal aging. The amount of erythrocyte-bound immunoglobulin G, the most direct and relevant parameter of erythrocyte aging, is significantly increased in Alzheimer patients relative to age-matched, healthy donors and to patients with multi-infarct dementia. These data indicate accelerated molecular breakdown of band 3 and premature appearance of senescent cell characteristics in erythrocytes from Alzheimer patients, and support the hypothesis that abnormal cellular aging may be involved in the etiology of the Alzheimer-specific pathology.

Erythrocyte membrane changes of individuals with Down's syndrome in various stages of Alzheimer-type dementia.

An increase in erythrocyte-bound IgG, enhanced breakdown of band 3, and changes in erythrocyte anion transport characteristics are observed in individuals with Down's Syndrome. We interpret these data as indicative for a disturbance of normal erythrocyte aging. A comparison of three groups of individuals with Down's Syndrome in various stages of dementia of the Alzheimer type did not reveal a correlation between the erythrocyte membrane changes and age or stage of dementia. Considering previously obtained data on patients with senile dementia of the Alzheimer type (7), these results may signify that parameters of disturbed erythrocyte aging represent presymptomatic markers of Alzheimer-type dementia.

Are thrombocyte membranes altered in Alzheimer's disease? A morphometric and biochemical study.

Morphometric analysis of thrombocytes from patients with Alzheimer's disease, from patients with multi-infarct dementia, and from young and age-matched healthy control donors, did not reveal any Alzheimer-related increase in internal membranes. Biochemical analysis showed a reduced cholesterol content of thrombocyte membrane preparations from Alzheimer patients relative to age-matched controls, but not relative to multi-infarct dementia patients. Overall distribution of protein kinase C activity (PKC) between cytosol and membrane, in resting as well as in activated thrombocytes from Alzheimer patients, was similar to that in the control groups. However, both Alzheimer and multi-infarct dementia patients had lower cytosolic levels of basal kinase and PKC activities than age-matched controls, while only Alzheimer patients had lower cytoskeletal PKC activity than controls.

Erythrocyte aging characteristics in elderly individuals with beginning dementia.

An increase in erythrocyte-bound IgG and enhanced breakdown of the erythrocyte anion exchanger band 3, characteristics of normal erythrocyte aging are observed in old, healthy individuals when compared with young donors. These findings indicate that the rate of cellular aging increases with organismal aging. Results from previous studies on the same parameters have suggested that the erythrocyte aging process is disturbed in patients in advanced stages of Alzheimer type dementia, and in individuals with Down's syndrome who show no signs of dementia. In this study we find no changes in erythrocyte aging parameters in old individuals in beginning stages of dementia of various etiologies. We conclude that, in general, characteristics of disturbed erythrocyte aging cannot serve as presymptomatic markers of Alzheimer-type dementia.

Influence of aging and neurodegenerative disease on changes in band 3-like proteins in white blood cells.

Fluorescent microspheres were used to measure antibody-induced capping of leukocyte membrane proteins that are immunologically related to band 3, the anion exchanger of the erythrocyte. The degree of capping was found to increase with donor age. Surface labeling and capping characteristics of cells from healthy, age-matched controls were not different from those from patients with Alzheimer's disease, multi-infarct dementia, and Down's syndrome. Immunoblots, however, indicated increased expression and/or breakdown of band 3-like proteins in leukocytes from patients when compared with young and old control donors. These findings emphasize the possible involvement of band 3-like proteins of nucleated cells in aging and disease.

Erythrocyte aging in the demented elderly: a fluctuating process?

Measurement of erythrocyte aging parameters in patients with dementia indicates that an Alzheimer-related disturbance of the erythrocyte aging process may not be detectable until in the more advanced stages of the disease. Also, a strong fluctuation in the values of erythrocyte aging parameters, over a period of 15 months, was observed in patients with dementia, but not in age-matched control donors. This fluctuation was independent of the type and stage of dementia, and its cause remains to be elucidated. Such variability hampers the use of erythrocyte aging characteristics for the diagnosis of dementia. On the other hand, the aging-related erythrocyte IgG content may be a sensitive biomarker for disturbed systemic homeostasis in the elderly.

No evidence for reduced thrombocyte cytochrome oxidase activity in Alzheimer's disease.

We measured cytochrome oxidase activity in thrombocytes from patients with senile dementia of the Alzheimer type. We found no decrease in enzyme activity in Alzheimer's disease patients when compared with patients with multi-infarct dementia, with young control donors, and with age-matched control donors.

Expression of the anion exchanger (AE) gene family in human brain. Identification of a new AE protein: AE0.

We present the first identification of proteins of the anion exchanger (AE) gene family expressed in human brain. Expression was established by the reverse transcriptase-polymerase chain reaction (RT-PCR), performed on RNA isolated from frontal cortex tissue. The erythroid form AE1, the major non-erythroid form AE2, and a novel member of the AE family, which we named AE0, were identified. Immunohistochemical analysis revealed the highest expression of these proteins in large pyramidal neurons in the frontal cortex and hippocampus. The sequence of the membrane domain of AE0 is identical to that of the major erythroid species AE1, except for the third extracellular loop, which contains a 25 amino acid insertion. This insertion is identical to a sequence in the third extracellular loop of AE2. Expression of AE0 is not restricted to brain tissue, since we could also detect AE0-mRNA in T-lymphocytes and reticulocytes. Chromosomal mapping indicates that the AE0 gene is most likely located on human chromosome 22. We did not find any indications for qualitative changes in AE1, AE2, or AE0 in Alzheimer brain tissue.

Anion exchange proteins are a component of corpora amylacea in Alzheimer disease brain.

Corpora amylacea (CAm) are amorphous structures that increase in number in the brains of patients with neurodegenerative diseases. We found abundant CAm in the brains of Alzheimer disease (AD) patients. We show here that proteins of the anion exchanger (AE) gene family, related to erythrocyte band 3, are present in CAm. The presence of AE epitopes in CAm may be related to the presence of AE proteins in membranes of neurones, and their altered expression level in degenerating neurones in AD brains. Whereas the distribution of immunoreactivity was uniform over the CAm in control brains, there was much more variation in staining of CAm core and peripheral structures in AD brains. Our data support the suggestion that accumulation of altered neuronal membrane proteins may be involved in the pathogenesis of CAm in AD.

Increased expression of heat-shock protein 27 kDa in Alzheimer disease: a preliminary study.

Stress-response (heat shock) proteins (hsps) are induced in living cells under pathological conditions, including diseases of the central nervous system. Increased synthesis of hsps is suggested to play a role in preventing neuronal injury in Alzheimer disease (AD). Using a highly specific antibody we have studied the expression of heat shock protein 27 kDa (hsp 27), in the brains of AD and non-demented, age-matched control patients. Immunoblotting and immunohistochemical methods were used. We report here that in the human brain in the normal condition the expression of hsp 27 is low and limited to the vessels, subpial astrocytes and single astrocytes of the white matter. There is a significant increase in the expression of hsp 27 in the cortex of AD. In AD, the immunoreaction is mainly localized in proliferating astrocytes establishing a pattern of astrocytic gliosis. These findings are the first to show the presence of hsp 27 in human cerebral tissue in normal conditions and its induction in AD.

Dementia, gliosis and expression of the small heat shock proteins hsp27 and alpha B-crystallin in Parkinson's disease.

Cognitive impairment and dementia are common in the later stages of Parkinson's disease (PD). Neuropathological examination of demented PD (PDD) patients often reveals changes that are typical of Alzheimer's disease (AD). In AD, there is a massive reactive gliosis and increased expression of the small heat shock proteins (hsp) hsp27 and alpha B-crystallin. Since these proteins are characteristic for reactive astrocytes in AD, we investigated their expression in the brains of PDD patients. The results were compared with those obtained in the brains of non-demented PD patients. We found (1) no detectable expression of hsp in PD without dementia, and low expression in PD with mild dementia; (2) reactive gliosis and increased expression of hsp in the cortex of PDD brains; (3) a strong association between hsp immunoreactivity and the severity of the AD-specific changes, especially with the number of tangles in the hippocampus; (4) a distinct immunoreaction of alpha B-crystallin in microglia in the substantia nigra and in the hippocampus in PDD. These results indicate that astrocytes react to the disease conditions in AD and in PDD in a similar way, namely by the increased expression of small heat shock proteins, and present additional evidence for the thesis that the pathology of the dementia in PD is related to that in AD.

Heat shock, but not the reactive state per se, induces increased expression of the small stress proteins hsp25 and alpha B-crystallin in glial cells in vitro.

The stress proteins hsp25 and alpha B-crystallin are found in increased concentrations in reactive astrocytes of brains undergoing neurodegeneration. In order to characterize this reaction, we investigated the expression of hsp25 and alpha B-crystallin during growth and after stress (heat shock) in glial cells in vitro. In primary rat brain cultures, hsp25 was present in actively dividing astrocytes that were positive for glial fibrillary acidic protein. alpha B-crystallin was found predominantly in oligodendrocytes. Heat shock resulted in increased concentrations of hsp25 and alpha B-crystallin in astrocytes, without any detectable changes in intracellular localization, as detectable with confocal laser microscopy. These results indicate that a neurodegeneration-related increase of the small stress proteins in astrocytes in independent of gliosis per se, and may be a disease-related event.

Anion exchange proteins and regulation of intracellular pH in cultured rat astrocytes and neurones.

We used primary cultures of rat hippocampal tissue to estimate the contribution of anion exchange (AE) proteins to the regulation of intracellular pH in neurones and astrocytes. After induction of acidosis, neonatal rat astrocytes were able to restore the intracellular pH in the absence of extracellular bicarbonate. Neonatal neurones, however, were able to recover from acidosis only when bicarbonate was present in the extracellular medium. This recovery was inhibited by inhibition of anion exchange and was independent of the presence of sodium ions. Antibodies against AE proteins reacted predominantly with neurones. These data suggest that neurones in particular are dependent on functional AE proteins for the maintenance of their intracellular pH.