Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS

Visokotlačna tekućinska kromatografija s detektorom raspršenja svjetla u uparenom uzorku za određivanje količine glavnih razreda fosfolipida u magarećem serumu

An HPLC method with an evaporative laser scattering detector was used to quantify major phospholipids fractions in donkeys’ serum. Blood samples were collected bimonthly for a whole year from 20 donkeys (10 male and 10 female) from the Sudanese breed kept at the premises of the Central Veterinary Research Laboratory (CVRL), Soba. The method used made the excellent separation possible of phosphatidylglycerine (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC), and sphingomyelin (SM). in 27 minutes, including the regeneration of the column. The SM resulted in two peaks (saturated and unsaturated fatty acids), as described by other researchers. The method was unable to separate phosphatidylserine (PS), which appeared in one peak with phosphatidylinositol. There is a signififi cant difference in the level of PG, PE, PI and SM between females and males.The method fully discussed in this study and the obtained values of major phospholipids can be used for health control and diagnosis of diseases.Visokotlačna tekućinska kromatografija s detektorom raspršenja svjetla u uparenom uzorku rabljena je za određivanje količine glavnih frakcija fosfolipida u magarećem serumu. Uzorci krvi bili su uzeti od 20 životinja (10 magaraca i 10 magarica sudanske pasmine) držanih u prostorima Središnjega veterinarskoga istraživačkoga laboratorija, Soba. Rabljena metoda omogućila je izvrsno odvajanje fosfatidilglicerola (FG), fosfatidiletanolamina (FE), fosfatidilinozitola (FI), fosfatidilkolina i sfifi ngomijelina (SM) za 27 minuta uključujući i regeneraciju kolone. Sfingomijelin je pokazivao dva vrška (zasićene i nezasićene masne kiseline) kao što je opisano od drugih autora. Rabljenom metodom nije se moglo odvojiti fosfatidilserin od fosfatidilinozitola. Ustanovljena je signifikantna razlika u razini FG, FE, FI i SM u mužjaka i ženki. U radu se iscrpno raspravlja o metodi, a dobivene vrijednosti glavnih fosfolipida mogu se upotrijebiti za kontrolu zdravlja i dijagnosticiranje bolesti

Trafficking pathways of Cx49-GFP in living mammalian cells

In the present study we examined the trafficking pathways of connexin49 (Cx49) fused to green fluorescent protein (GFP) in polar and non-polar cell lines. The Cx49 gene was isolated from ovine lens by RT-PCR. Cx49 cDNA was fused to GFP and the hybrid cDNA was transfected into several cell lines. After transfection of Cx49-GFP cDNA into HeLa cells, it was shown using the double whole-cell patch-clamp technique that the expressed fusion protein was still able to form conducting gap junction channels. Synthesis, assembly, and turnover of the Cx49-GFP hybrid protein were investigated using a pulse-chase protocol. A major 78-kDa protein band corresponding to Cx49-GFP could be detected with a turnover of 16-20 h and a half-life time of 10 h. The trafficking pathways of Cx49-GFP were monitored by confocal laser microscopy. Fusion proteins were localized in subcellular compartments, including the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment, the Golgi apparatus, and the trans-Golgi network, as well as vesicles traveling towards the plasma membrane. Time-dependent sequential localization of Cx49-GFP in the ER and then the Golgi apparatus supports the notion of a slow turnover of Cx49-GFP compared to other connexins analyzed so far. Gap junction plaques resembling the usual punctuate distribution pattern could be demonstrated for COS-1 and MDCK cells. Basolateral distribution of Cx49-GFP was observed in polar MDCK cells, indicating specific sorting behavior of Cx49 in polarized cells. Together, this report describes the first characterization of biosynthesis and trafficking of lens Cx49.Fritz Thyssen-Stiftun

Ex vivo biochemical analysis of CFTR in human rectal biopsies

AbstractThis report describes the first biosynthetic analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) in freshly excised human rectal biopsies. Expression of functional CFTR was assessed by intestinal current measurement (ICM) prior to biosynthetic studies. Several structural features of CFTR are found to be comparable to those established in CFTR-expressing cell lines. Interestingly, maturation of CFTR increases substantially in tissue incubated at 26 °C. Our data provide a solid basis for future studies on the characterisation of CFTR in pathological cases

Striking structural and functional similarities suggest that intestinal sucrase-isomaltase, human lysosomal α-glucosidase and Schwanniomyces occidentalis glucoamylase are derived from a common ancestral gene

AbstractSequence comparison of the primary structure of the yeast Schwanniomyces occidentalis glucoamylase (GAM) with GAMs in different microorganisms did not reveal significant similarities. By contrast, striking similarities were, surprisingly, found with 3 mammalian secretory and integral membrane proteins: the 2 subunits of intestinal brush border sucrase-isomaltase and human lysosomal α-glucosidase. The similarities among these proteins are found as clusters of up to 8 amino acids and distributed all over the protein sequences. The major sequence differences are found in the N-terminal regions accounting, probably, for the different cellular locations of these proteins. The high level of similarities between sucrase, isomaltase, Sch. occidentalis GAM and human lysosomal α-glucosidase suggest that these proteins are derived from the same ancestral gene. To our knowledge, this is the first report that describes similarities between a yeast secretory protein and mammalian secretory and integral membrane proteins

Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing.

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA

Competitive random sequential adsorption of point and fixed-sized particles: analytical results

We study the kinetics of competitive random sequential adsorption (RSA) of particles of binary mixture of points and fixed-sized particles within the mean-field approach. The present work is a generalization of the random car parking problem in the sense that it considers the case when either a car of fixed size is parked with probability q or the parking space is partitioned into two smaller spaces with probability (1-q) at each time event. This allows an interesting interplay between the classical RSA problem at one extreme (q=1), and the kinetics of fragmentation processes at the other extreme (q=0). We present exact analytical results for coverage for a whole range of q values, and physical explanations are given for different aspects of the problem. In addition, a comprehensive account of the scaling theory, emphasizing on dimensional analysis, is presented, and the exact expression for the scaling function and exponents are obtained.Comment: 7 pages, latex, 3 figure

Case study on the pathophysiology of Fabry disease: abnormalities of cellular membranes can be reversed by substrate reduction in vitro

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with N-butyldeoxynojirimycin (NB-DNJ) in vitro was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients

Genetic reporter analysis reveals an expandable reservoir of OCT4+ cells in adult skin

The transcription factor Oct4 (Pou5f1) is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells. Therefore, Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming. However, analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms. Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4+ cells in postnatal mice. While the prevalence of GFP+ cells in testis rapidly declined with age, the skin-resident GFP+ population expanded in a cyclical fashion. These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle, which endogenously expressed all principle reprogramming factors at low levels. Interestingly, skin wounding or non-traumatic hair removal robustly expanded the GFP+ epidermal cell pool not only locally, but also in uninjured skin areas, demonstrating the existence of a systemic response. Thus, the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4+ stem cells, which might be useful for therapeutic cell transfer or facilitated reprogramming. Electronic supplementary material The online version of this article (doi: 10.1186/2045-9769-3-9) contains supplementary material, which is available to authorized users

Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology