An investigation of the effects of antitumour and other drugs on cell morphology and the cytoskeleton of erythrocytes

Human arythrocytes were used as a model system for an investigation of the mechanism of action of the antiproliferative drug Adriamycin. Erythrocytes were induced to undergo a change in morphology by elevation of intracellular calcium. It was revealed that the widely used media employed for the study of morphological change were unsuitable; a new incubation medium was developed so that cells were metabolically replete. In this medium echinocytosis took place both in a calcium concentration- and time-dependent manner. Pretreatment of erythrocytes with Adriamycin (10 M for 10 mins) protected the erythrocytes against calcium-induced echinocytosis at calcium concentrations < 150M. SDS-PAGE analysis of the cytoskeletal proteins prepared from erythrocytes revealed the calcium-induced proteolysis of two main cytoskeletal proteins: band 2:1 and band 4:1. Only the rate of the proteolysis of band 2.1 correlated with the onset of echinocytosis. Adriamycin inhibited the breakdown of band 2.1 even when the cells formed echinocytes; this raises doubts concerning the importance of band 2.1 in the maintenance of discocyte morphology. Adriamycin only marginally inhibited the purified calcium-activated thio protease (calpain). Calcium-loading of human erythrocytes increased the phosphorylation of several major cytoskeletal proteins including pp120, band 3, band 4.1 and band 4.9. The pattern of increase resembled that induced by 12-0-tetradecanoyl-phorbol-13-acetate. Pre-treatment with Adriamycin prior to calcium loading caused a general lowering of basal phosphorylation. Adriamycin had no effect on the activity of the calcium-activated phospholipid-dependent protein kinase (protein kinase C). A hypothesis is put forward that the morphological transition of erythrocytes might be dependent upon the activity of a contractile system.

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.

Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

Improper organization of the actin cytoskeleton affects protein synthesis at initiation

Although the actin cytoskeleton and the translation machinery are considered to be separate cellular complexes, growing evidence supports overlapping regulation of the two systems. Because of its interaction with actin, the eukaryotic translation elongation factor 1A (eEF1A) is proposed to be a regulator or link between these processes. Using a genetic approach with the yeast Saccharomyces cerevisiae, specific regions of eEF1A responsible for actin interactions and bundling were identified. Five new mutations were identified along one face of eEF1A. Dramatic changes in cell growth, cell morphology, and actin cable and patch formation as well as a unique effect on total translation in strains expressing the F308L or S405P eEF1A mutant form were observed. The translation effects do not correlate with reduced translation elongation but instead include an initiation defect. Biochemical analysis of the eEF1A mutant forms demonstrated reduced actin-bundling activity in vitro. Reduced total translation and/or the accumulation of 80S ribosomes in strains with either a mutation or a null allele of genes encoding actin itself or actin-regulating proteins Tpm1p, Mdm20p, and Bnirp/Bni1p was observed. Our data demonstrate that eEF1A, other actin binding proteins, and actin mutants affect translation initiation through the actin cytoskeleton.

What does the study of the spatial patterns of pathological lesions tell us about the pathogenesis of neurodegenerative disorders?

Discrete pathological lesions, which include extracellular protein deposits, intracellular inclusions and changes in cell morphology, occur in the brain in the majority of neurodegenerative disorders. These lesions are not randomly distributed in the brain but exhibit a spatial pattern, that is, a departure from randomness towards regularity or clustering. The spatial pattern of a lesion may reflect pathological processes affecting particular neuroanatomical structures and, therefore, studies of spatial pattern may help to elucidate the pathogenesis of a lesion and of the disorders themselves. The present article reviews first, the statistical methods used to detect spatial patterns and second, the types of spatial patterns exhibited by pathological lesions in a variety of disorders which include Alzheimer's disease, Down syndrome, dementia with Lewy bodies, Creutzfeldt-Jakob disease, Pick's disease and corticobasal degeneration. These studies suggest that despite the morphological and molecular diversity of brain lesions, they often exhibit a common type of spatial pattern (i.e. aggregation into clusters that are regularly distributed in the tissue). The pathogenic implications of spatial pattern analysis are discussed with reference to the individual disorders and to studies of neurodegeneration as a whole.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Effects of oxidative stress and neuroprotection in apoptosis in neuronal cell models

The PC12 and SH-SY5Y cell models have been proposed as potentially realistic models to investigate neuronal cell toxicity. The effects of oxidative stress (OS) caused by both H2O2 and Aβ on both cell models were assessed by several methods. Cell toxicity was quantitated by measuring cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) viability assay, an indicator of the integrity of the electron transfer chain (ETC), and cell morphology by fluorescence and video microscopy, both of which showed OS to cause decreased viability and changes in morphology. Levels of intracellular peroxide production, and changes in glutathione and carbonyl levels were also assessed, which showed OS to cause increases in intracellular peroxide production, glutathione and carbonyl levels. Differentiated SH-SY5y cells were also employed and observed to exhibit the greatest sensitivity to toxicity. The neurotrophic factor, nerve growth factor (NGF) was shown to cause protection against OS. Cells pre-treated with NGF showed higher viability after OS, generally less apoptotic morphology, recorded less apoptotic nucleiods, generally lower levels of intracellular peroxides and changes in gene expression. The neutrophic factor, brain derived growth factor (BDNF) and ascorbic acid (AA) were also investigated. BDNF showed no specific neuroprotection, however the preliminary data does warrant further investigation. AA showed a 'janus face' showing either anti-oxidant action and neuroprotection or pro-oxidant action depending on the situation. Results showed that the toxic effects of compounds such as Aβ and H2O2 are cell type dependent, and that OS alters glutathione metabolism in neuronal cells. Following toxic insult, glutathione levels are depleted to low levels. It is herein suggested that this lowering triggers an adaptive response causing alterations in glutathione metabolism as assessed by evaluation of glutathione mRNA biosynthetic enzyme expression and the subsequent increase in glutathione peroxidase (GPX) levels.

Insulin gene therapy for the treatment of diabetes mellitus

Currently available treatments for insulin-dependent diabetes mellitus are often inadequate in terms of both efficacy and patient compliance. Gene therapy offers the possibility of a novel and improved method by which exogenous insulin can be delivered to a patient. This was approached in the present study by constructing a novel insulin-secreting cell line. For the purposes of this work immortalized cell lines were used. Fibroblasts and pituitary cells were transfected with the human preproisinulin gene to create stable lines of proinsulin- and insulin-secreting cells. The effect of known β-cell secretagogues on these cells were investigated, and found mostly to have no stimulatory effect, although IBMX, arginine and ZnSO4 each increased the rate of secretion. Cyclosporin (CyA) is currently the immunosuppresant of choice for transplant recipients; the effect of this treatment on endogenous β-cell function was assessed both in vivo and in vitro. Therapeutic doses of CyA were found to reduce plasma insulin concentrations and to impair glucose tolerance. The effect of immunoisolation on insulin release by HIT T15 cells was also investigated. The presence of an alginate membrane was found to severely impair insulin release. For the first implantation of the insulin-secreting cells, the animal model selected was the athymic nude mouse. This animal is immunoincompetent, and hence the use of an immunosuppressive regimen is circumvented. Graft function was assessed by measurement of plasma human C peptide concentrations, using a highly specific assay. Intraperitoneal implantation of genetically manipulated insulin-secreting pituitary cells into nude mice subsequently treated with a large dose of streptozotocin (STZ) resulted in a significantly delayed onset of hyperglycaemia when compared to control animals. Consumption of a ZnSO4 solution was shown to increase human C peptide release by the implant. Ensuing studies in nude mice examined the efficacy of different implantation sites, and included histochemical examination of the tumours. Aldehyde fuchsin staining and immunocytochemical processing demonstrated the presence of insulin containing cells within the excised tissue. Following initial investigations in nude mice, implantation studies were performed in CyA-immunosuppressed normal and STZ-diabetic mice. Graft function was found to be less efficacious, possibly due to the subcutaneous implantation site, or to the immunosuppresive regimen. Histochemical and transmission electron microscopic analysis of the tumour-like cell clusters found at autopsy revealed necrosis of cells at the core, but essentially normal cell morphology, with dense secretory granules in peripheral cells. The thesis provides evidence that gene therapy offers a feasibly new approach to insulin delivery.

Characterization of human mesenchymal stem cells from multiple donors and the implications for large scale bioprocess development

Cell-based therapies have the potential to contribute to global healthcare, whereby the use of living cells and tissues can be used as medicinal therapies. Despite this potential, many challenges remain before the full value of this emerging field can be realized. The characterization of input material for cell-based therapy bioprocesses from multiple donors is necessary to identify and understand the potential implications of input variation on process development. In this work, we have characterized bone marrow derived human mesenchymal stem cells (BM-hMSCs) from multiple donors and discussed the implications of the measurable input variation on the development of autologous and allogeneic cell-based therapy manufacturing processes. The range of cumulative population doublings across the five BM-hMSC lines over 30 days of culture was 5.93, with an 18.2% range in colony forming efficiency at the end of the culture process and a 55.1% difference in the production of interleukin-6 between these cell lines. It has been demonstrated that this variation results in a range in the process time between these donor hMSC lines for a hypothetical product of over 13 days, creating potential batch timing issues when manufacturing products from multiple patients. All BM-hMSC donor lines demonstrated conformity to the ISCT criteria but showed a difference in cell morphology. Metabolite analysis showed that hMSCs from the different donors have a range in glucose consumption of 26.98 pmol cell−1 day−1, Lactate production of 29.45 pmol cell−1 day−1 and ammonium production of 1.35 pmol cell−1 day−1, demonstrating the extent of donor variability throughout the expansion process. Measuring informative product attributes during process development will facilitate progress towards consistent manufacturing processes, a critical step in the translation cell-based therapies.

Overlap between neurodegenerative disorders

Neurodegenerative disorders are characterized by the formation of distinct pathological changes in the brain, including extracellular protein deposits, cellular inclusions, and changes in cell morphology. Since the earliest published descriptions of these disorders, diagnosis has been based on clinicopathological features, namely, the coexistence of a specific clinical profile together with the presence or absence of particular types of lesion. In addition, the molecular profile of lesions has become an increasingly important feature both in the diagnosis of existing disorders and in the description of new disease entities. Recent studies, however, have reported considerable overlap between the clinicopathological features of many disorders leading to difficulties in the diagnosis of individual cases and to calls for a new classification of neurodegenerative disease. This article discusses: (i) the nature and degree of the overlap between different neurodegenerative disorders and includes a discussion of Alzheimer's disease, dementia with Lewy bodies, the fronto-temporal dementias, and prion disease; (ii) the factors that contribute to disease overlap, including historical factors, the presence of disease heterogeneity, age-related changes, the problem of apolipoprotein genotype, and the co-occurrence of common diseases; and (iii) whether the current nosological status of disorders should be reconsidered.

Are pathological lesions in neurodegenerative disorders the cause or the effect of the degeneration?

Pathological lesions in the form of extracellular protein deposits, intracellular inclusions and changes in cell morphology occur in the brain in the majority of neurodegenerative disorders. Studies of the presence, distribution, and molecular determinants of these lesions are often used to define individual disorders and to establish the mechanisms of lesion pathogenesis. In most disorders, however, the relationship between the appearance of a lesion and the underlying disease process is unclear. Two hypotheses are proposed which could explain this relationship: (i) lesions are the direct cause of the observed neurodegeneration ('causal' hypothesis); and (ii) lesions are a reaction to neurodegeneration ('reaction' hypothesis). These hypotheses are considered in relation to studies of the morphology and molecular determinants of lesions, the effects of gene mutations, degeneration induced by head injury, the effects of experimentally induced brain lesions, transgenic studies and the degeneration of anatomical pathways. The balance of evidence suggests that in many disorders, the appearance of the pathological lesions is a reaction to degenerative processes rather than being their cause. Such a conclusion has implications both for the classification of neurodegenerative disorders and for studies of disease pathogenesis.

Unexpected role of surface transglutaminase type II in celiac disease

Background & Aims: In celiac disease (CD), transglutaminase type II (TG2) has 2 fundamental roles: (1) as the autoantigen recognized by highly specific autoantibodies and (2) the modifier of pathogenic gliadin T-cell epitopes. It follows that inhibition of TG2 might represent an attractive strategy to curb the toxic action of gliadin. Here we studied the validity of this strategy using the organ culture approach. Methods: Duodenal biopsy specimens from 30 treated patients with CD, 33 untreated patients with CD, and 24 controls were cultured with or without gliadin peptides p31-43, pα-9, and deamidated pα-9 for 20 minutes, 3 hours, and 24 hours. In 31 patients with CD and 16 controls, TG2 inhibitor R283 or anti-TG CUB 7402 or anti-surface TG2 (6B9) mAbs were used in cultures. T84 cells were also cultured with or without peptides with or without TG inhibitors. Mucosal modifications after culture were assessed by immunofluorescence, in situ detection of TG activity, confocal microscopy, and fluorescence-activated cell sorter analysis. Results: The enzymatic inhibition of TG2 only controlled gliadin-specific T-cell activation. The binding of surface TG2 contained gliadin-specific T-cell activation and p31-43-induced actin rearrangement, epithelial phosphorylation, and apoptosis, both in organ cultures and T84 cells. Conclusions: These data indicate a novel and unexpected biological role for surface TG2 in the pathogenesis of CD suggesting a third role for TG2 in CD. These results have a specific impact for celiac disease, with wider implications indicating a novel biologic function of TG2 with possible repercussions in other diseases. © 2005 by the American Gastroenterological Association.

The postantibiotic effect of the carbapenem antibiotics on gram-negative bacteria

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.