Boar spermatozoa successfully predict mitochondrial modes of toxicity: implications for drug toxicity testing and the 3R principles.

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker & JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P<0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r=0.559) and proportions of motile (r=0.55) or progressively motile (r=0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

The effects of oats on the function of gut microflora in children with coeliac disease.

BACKGROUND: Faecal short chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported high faecal SCFA levels in children with coeliac disease (CD), indicating alteration in gut microfloral metabolism. Data accumulated over recent decades by us and others suggest that wheat-free oats can safely be included in a gluten-free diet (GFD). However, concerns have been raised with respect to the safety of oats in a subset of coeliacs. AIM: To describe faecal SCFA patterns in children with newly diagnosed CD treated for 1 year with a GFD with or without oats. METHODS: This report is part of a randomised, double-blind study on the effect of a GFD containing oats (GFD-oats) vs. a standard GFD (GFD-std). Faecal samples were received from 34 children in the GFD-oats group and 37 in the GFD-std group at initial diagnosis and/or after 1 year on a GFD. Faecal SCFAs were analysed. RESULTS: The GFD-std group had a significantly lower total faecal SCFA concentration at 12 months compared with 0 months (P < 0.05). In contrast, total SCFA in the GFD-oats group remained high after 1 year on the GFD. The children in the GFD-oats group had significantly higher acetic acid (P < 0.05), n-butyric acid (P < 0.05) and total SCFA concentration (P < 0.01) after 1-year diet treatment compared to the GFD-std group. CONCLUSIONS: Our results indicate that oats do affect the gut microflora function, and that some coeliac children receiving oats may develop gut mucosal inflammation, that may present a risk for future complications.

[Novel splicing isoform of actin-binding protein alpha-actinin 4 in epidermoid carcinoma cells A431].

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

Low levels of bile acids increase bacterial uptake in colonic biopsies from patients with collagenous colitis in remission.

BACKGROUND: Patients with collagenous colitis have an impaired mucosal barrier. Moreover, collagenous colitis is associated with bile acid malabsorption. Bile acids can increase bacterial mucosal uptake in humans. Mucosal barrier function was investigated by exposing colonic biopsies to chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) in Ussing chamber experiments. AIM: To find if low levels of bile acids increase bacterial uptake in colonic biopsies from collagenous colitis patients. METHODS: The study comprised 33 individuals; 25 with collagenous colitis (14 in clinical remission without treatment, 11 with active disease and 10 examined in clinical remission resulting from treatment with 6 mg budesonide); eight healthy individuals undergoing screening colonoscopy served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short-circuit current (Isc), potential difference, trans-epithelial resistance and transmucosal passage of Escherichia coli K12 after adding 100 µmol/L CDCA or DCA. RESULTS: When adding 100 µmol/L CDCA or DCA, bacterial uptake increased fourfold in biopsies of patients in remission; CDCA 6.5 units [2.5-9.8] and DCA 6.2 units [2.1-22] (median [IQR]), compared with uptake in biopsies without added bile acids 1.6 units [1.1-3] (P=0.004 and P=0.01 respectively). In active disease and in patients in remission due to budesonide treatment, bile acids did not affect bacterial uptake. Confocal microscopy revealed trans-epithelial passage of E. coli K12 within 30 min. CONCLUSIONS: Low concentrations of dihydroxy-bile acids exacerbate mucosal barrier dysfunction in colonic biopsies of patients with collagenous colitis in remission. This allows a substantially increased bacterial uptake, which may contribute to recurrence of inflammation.

Children with screening-detected coeliac disease show increased levels of nitric oxide products in urine.

AIM: Increased concentration of nitric oxide (NO) metabolites, nitrite and nitrate, in the urine is a strong indication of ongoing small intestinal inflammation, which is a hallmark of the enteropathy of coeliac disease (CD). It has previously been shown that children with symptomatic, untreated CD have increased levels of NO oxidation products in their urine. The aim of this study was to investigate whether screening-detected, asymptomatic coeliac children display the same urinary nitrite/nitrate pattern. METHODS: In a multicenter screening study, serum samples were collected from 7208 12-year-old children without previously diagnosed CD. Sera were analysed for anti-human tissue transglutaminase (tTG) of isotype IgA. Small bowel biopsy was performed in antibody-positive children, yielding 153 new cases of CD. In the screening-detected individuals, the sum of nitrite and nitrate concentrations in the urine was analysed and used as an indicator of NO production. For comparison, 73 children with untreated, symptomatic CD were studied. RESULTS: The nitrite/nitrate levels in children with screening-detected CD and those with untreated symptomatic CD did not differ significantly. Both groups had significantly increased urinary nitrite/nitrate concentrations compared to the children with normal small bowel biopsy (p < 0.001). CONCLUSION: Children with screening-detected CD have increased production of NO just as children with untreated symptomatic CD. High NO metabolite levels in the urine may indicate a pathogenetic feature of CD and be a marker of major clinical importance.

α-Dystrobrevin distribution and association with other proteins in human promyelocytic NB4 cells treated for granulocytic differentiation.

Dystrobrevins (DBs) bind directly to dystrophin and are prominent components of the dystrophin-associated protein complex (DAPC) that links the cytoskeleton to the extracellular matrix. They are involved in brain development, synapse formation and plasticity, as well as water and ion homeostasis. However, the role of DB in non-muscular cells is not clear. In this study, we show that different α-dystrobrevin isoforms are present in promyelocytic leukemia (NB4) cells. Only the biggest α-dystrobrevin isoform (DB-α), which can be important for its function, was expressed in the membrane fraction of NB4 cells; the other α-DB isoforms were found in the hydrophilic cell fractions. Employing the immunoprecipitation and mass spectrometry, we identified novel α-DB-interacting proteins involved in cytoskeleton reorganization (actin, tropomyosin, gelsolin, tubulin) and signal transduction process (stathmin, prohibitin, RIBA) during proliferation and differentiation of NB4 cells. Our results suggest that α-DB isoforms play a central role in cytoskeleton reorganization via their multiple interactions with actin and actin-associating proteins and may participate in signal transduction process during NB4 cell granulocytic differentiation via directly and non directly associated proteins.

Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.

Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10(6) kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.

[Analysis of nuclear protein complexes comprising alpha-actinin-4 by 2D-electrophoresis and mass-spectrometry].

Actin-binding protein alpha-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of alpha-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with alpha-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear alpha-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. beta-Actin, alpha- and beta-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with beta-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with alpha-actinin-4 may suggest that alpha-actinin-4 is involved in transcription and splicing. Presence of beta-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDI-TOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against alpha-tubulin confirmed association of alpha-actinin-4 with alpha-tubulin in the protein complex. Nuclear alpha-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with alpha-tubulin. These data suggest that alpha-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.

[Analysis of extracellular matrix proteins produced by cultured cells].

Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.

[The comparative analysis of subcellular fractionation methods for revealing of alpha-actinin 1 and alpha-actinin 4 in A431 cells].

Alpha-actinin 1 and alpha-actinin 4 belong to a family of actin-binding proteins with shared structural function and regulation of several processes in a cell. Based on previous data on different distribution of these proteins in the nucleus and cytoplasm, we have explored in detail the distribution of alpha-actinin 1 and alpha-actinin 4 in subcellular fractions in A431 cells spread on fibronectin. Several methods of subcellular fractionation were used. Complex approach allowed resuming that revealing of alpha-actinin isoforms in fractions depended on the composition of lysis buffer and preliminary low-temperature freezing of the cells. We have drawn a conclusion that alpha-actinin 4 can be found in all cytoplasmic and nuclear subfractions, while alpha-actinin 1 is characterized by cytoplasmic and membrane localization with specificity of its distribution tightly to the nuclear membrane.

Increased uptake of non-pathogenic E. coli via the follicle-associated epithelium in longstanding ileal Crohn's disease.

In Crohn's disease (CD), inflammation is driven by luminal commensal micro-organisms; however, mechanisms of early phases of inflammation need further clarification. The earliest observable lesions of recurrent CD are microscopic erosions at the specialized follicle-associated epithelium (FAE), which lines the Peyer's patches. Therefore, our aim was to investigate the mucosal barrier to non-pathogenic bacteria in FAE of CD. The FAE of macroscopically normal ileum from patients with longstanding CD, ulcerative colitis, and controls was studied in Ussing chambers regarding electrophysiology and permeability to 51Cr-EDTA, horseradish peroxidase, and non-pathogenic E. coli strains. Transepithelial passage routes and uptake into dendritic cells were studied by confocal and electron microscopy. FAE of CD showed increased numbers of adherent bacteria, after E. coli exposure in Ussing chambers, as well as spontaneously in non-exposed archival surgical tissues. Further, we found increased uptake of fluorescent E. coli K-12 and HB101 across FAE of CD, but not in ulcerative colitis. Microscopy demonstrated intercellular and transcellular uptake of E. coli in CD, but only transcellular in controls. FAE exposed to E. coli demonstrated changes in conductance and 51Cr-EDTA permeability, suggesting that bacteria affected the paracellular pathway in CD mucosa. Following bacterial uptake, CD mucosa also demonstrated an increased percentage of E. coli co-localizing with dendritic cells, and augmented tissue release of TNF-alpha. Our data present novel insights into the pathophysiology of CD by demonstrating a previously unrecognized defect of FAE barrier to bacteria in ileal CD, leading to increased load of commensal bacteria to the inductive sites of mucosal immunity.

Bone marrow dendritic cells internalize live RF-81 bovine rotavirus and rotavirus-like particles (RF 2/6-GFP-VLP and RF 8*2/6/7-VLP) but are only activated by live bovine rotavirus.

Dendritic cells (DC) represent the link between innate and adaptive immunity. They are classified as antigen-presenting cells (APC) and can initiate and modulate the immune response. To investigate the interaction with DCs, live RF-81 bovine rotavirus strain (RFV) and rotavirus-like particles (rota-VLP), RF 2/6-GFP-VLP and rota RF 8*2/6/7-VLP, were added in vitro to murine bone marrow-derived DCs (bmDCs). Live RFV, RF 2/6-GFP-VLP and RF 8*2/6/7-VLP all bound to bmDC and were internalized but only live RFV stimulated phenotypic maturation of the bmDCs as shown by the upregulation of the co-stimulatory molecule CD86. Even though bmDCs internalized RF 2/6-GFP-VLP and RF 8*2/6/7-VLP as efficiently as live RFV, these rota-VLP were not able to activate the cells. Supernatants derived from bmDC cultures treated with live RFV, RF 2/6-GFP-VLP or RF 8*2/6/7-VLP were examined for TNF-alpha production. At 6, 18 and 24 h post-infection, TNF-alpha concentrations were significantly increased in cultures treated with live RFV and rota-VLP compared with untreated cultures. In conclusion, this study showed that live RF-81 bovine rotavirus strain was internalized and induced bmDCs activation, whereas both RF 2/6-GFP-VLP and RF 8*2/6/7-VLP were internalized by bmDCs without triggering their activation.

Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation.

Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase C alpha. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.

Association of alpha-dystrobrevin with reorganizing tight junctions.

Alpha-dystrobrevin (alpha-DB) has been described primarily as a cytoplasmic component of the dystrophin-glycoprotein complex in skeletal muscle cells. Isoforms of alpha-DB show different localization in cells and tissues; at basolateral membranes in epithelial cells, dystrobrevins mediate contact with the extracellular matrix, peripheral and transmembrane proteins and the filamentous actin cytoskeleton. Beside their structural role, alpha-DBs are assumed to be important in cell signalling and cell differentiation. We have primarily assessed the role of alpha-DB in two epithelial cell lines (MDCK I, HT 29), which represent different developmental stages and exhibit distinct permeability characteristics. Using a polyclonal anti-alpha-DB antibody, we have investigated its expression, localization and association with tight junction (TJ)- associated proteins (ZO-1, occludin) before and after protein kinase C (PKC) activation with phorbol myristate acetate. Distinct subsets of alpha-DB isoforms were detected in the two cell lines by immunoblotting. In both cell lines there was submembranous localization of alpha-DB both apically and basolaterally, shown with confocal imaging. PKC activation caused a reorganization of TJ, which was parallel to increased localization of alpha-DB to TJ areas, most pronounced in MDCK I cells. Moreover, actin and ZO-1 co-immunoprecipitated with a-DB, as displayed with immunoblotting. Our findings suggest that a-dystrobrevin specifically is associated with the tight junctions during their reorganization.

p21 (Waf1/Cip1) and FasL gene activation via Sp1 and NFkappaB is required for leukemia cell survival but not for cell death induced by diverse stimuli.

The molecular mechanisms of the cellular response to different apoptotic effectors are only partially understood. Herein, the role of transcription factors, Sp1 and NFkappaB in differentiation-related and etoposide-induced apoptosis was examined in a number of human leukemia cell lines (HL-60, NB4, HEL, THP-1, K562). This was investigated with respect to the recruitment of one cell-cycle regulating gene, p21 and one cell death gene, FasL. Using electrophoretic mobility shift assay (EMSA), we consistently observed Sp1 and NFkappaB binding activity to the promoter of either gene during cell differentiation and the decrease associated with apoptosis upon long-term treatment with differentiation inducers in HL-60, NB4 and HEL cells. By contrast, Sp1 and NFkappaB binding capacities were lost in all myeloid cell lines undergoing etoposide-induced fast apoptosis. This effect was eliminated by the broad-spectrum caspase inhibitor, benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone, thus restoring transcription factors' binding activity. However, sustained NFkappaB binding to the FasL promoter was noticed in apoptosis undergoing HEL cells treated by etoposide. Our results suggest that p21 and FasL gene activation is required for myeloid leukemia cell survival or maturation but not for cell death via Sp1 and NFkappaB as regulators of these genes. The findings also support the idea of a common mechanism for cellular responses to different apoptotic effectors in malignant hematopoietic cell lines.

Gut microflora associated characteristics in children with celiac disease.

OBJECTIVES: The aim of the study was to investigate the metabolic function of intestinal microflora in children with celiac disease (CD) in order to find out if there is a deviant gut flora in CD patients compared to healthy controls. METHODS: The study group comprised children with CD, consecutively diagnosed according to current criteria given by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition. Thirty-six children were studied at presentation, i.e., on a normal gluten-containing diet, with clinical symptoms and signs indicative of CD, positive celiac serology markers, and a small bowel biopsy showing severe enteropathy. Forty-seven patients were studied when they had been on a gluten-free diet (GFD) for at least 3 months. For comparison, a group of 42 healthy controls (HC) were studied. The functional status of the intestinal microflora was evaluated by gas-liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. RESULTS: There was a significant difference between untreated CD children and HC as well as between treated CD children and HC regarding acetic, i-butyric, i-valeric acid, and total SCFAs. The propionic and n-valeric acids differed significantly between CD children on GFD and HC. Moreover, there was a strong correlation between i-butyric and i-valeric acids in all study groups. CONCLUSIONS: This is the first study of the SCFA pattern in fecal samples from children with CD. The results indicate that there is a difference in the metabolic activity of intestinal microbial flora in children with CD compared to that in HC. The finding of a different pattern of some SCFAs in celiacs both at presentation and during treatment with GFD indicates that it is a genuine phenomenon of CD not affected by either the diet, the inflammation, or the autoimmune status of the patient.

Antibodies to oat prolamines (avenins) in children with coeliac disease.

BACKGROUND: The use of oats in a gluten-free diet for children with coeliac disease is presently under investigation. In this study we measured the content of antibodies to oat prolamines (avenin) in sera from coeliac children and reference children. METHODS: Crude avenin was prepared by extraction with ethanol and salt-solution and used as antigen in a three-step ELISA. Sera from 81 children, including 34 children with verified coeliac disease, were analysed for both IgA and IgG antibodies to avenin and gliadin. Sera were also incubated with gliadin before exposure to avenin, and vice versa, to assess a possible cross-reaction between the species. Keyhole limpet hemocyanin (KLH) was used as a negative control. RESULTS: Children with coeliac disease on a normal diet had significantly higher levels of antibodies to avenin, both IgG and IgA, than reference children (P < 0.001) and the levels correlated positively with gliadin antibodies, especially of IgA-type (r = 0.798). Both anti-avenin and anti-gliadin antibodies were only absorbed by the corresponding protein. CONCLUSIONS: Children with coeliac disease have antibodies to oat proteins at significantly higher levels than reference children. The absorption test did not indicate a cross-reactivity between the prolamines of wheat and oats. The method will be employed for repeated sampling of anti-avenin antibodies during a prospective interventional study with a gluten-free diet supplemented with oats.

Altered impedance during pigment aggregation in Xenopus laevis melanophores.

Melanophores are dark-brown pigment cells located in the skin of amphibia, fish and many invertebrates. The skin colour of these organisms is regulated by the translocation of pigment organelles, and the pigment distribution can be altered by external stimuli. The ability to change colour in response to stimuli makes these cells of interest for biosensing applications. It was investigated whether pigment aggregation in Xenopus laevis melanophores can be detected by impedance measurements performed in transparent microvials. The results show that cell attachment, cell spreading and pigment aggregation all resulted in impedance changes, seen particularly at the highest frequency tested (10 kHz). The mechanisms behind the impedance changes were investigated by the addition of latrunculin or melatonin, both of which cause pigment aggregation. The latrunculin-induced aggregation was associated with cell area decrease and filamentous actin (F-actin) breakdown, processes that can influence the impedance. Lack of F-actin breakdown and an increase in cell area during melatonin-induced aggregation suggest that some other intracellular process also contributes to the impedance decrease seen for melatonin. It was shown that impedance measurements reflect not only cell attachment and cell spreading, but also intracellular events.

Role of protein kinase C alpha for uptake of unopsonized prey and phagosomal maturation in macrophages.

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPG's effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.

Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins.

Phosphotyrosine signaling plays a vital role in cell regulation--from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphor lated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.