Western Blot Immunodetection Research Articles

Optimizing Western blotting immunodetection: Streamlining antibody cocktails for reduced protocol time and enhanced multiplexing applications.

Adaptive, rather than innate, immunity relies mainly on antigen-antibody recognition. This recognition is driven by the binding of specific antibody paratopes to distinct epitopes found on antigens. This interaction is pivotal for immune responses that have been re-purposed for diagnostic and therapeutic purposes. This article focuses on Western blotting, an in vitro technique performed for protein immunodetection. Traditionally, this technique requires separate incubations of both primary and secondary antibodies, for which these antibodies recognize different antigen epitopes (conventional method). We propose a modified protocol combining both antibodies, involving a single incubation step that reduces time and conserves reagents (non-conventional/improved method). This improved protocol will enhance efficiency without compromising detection accuracy. It will support multiplexing, enabling the simultaneous detection of multiple proteins. Despite the positive results found by applying available antibodies, further optimization is required for a more thorough evaluation, to ensure that all antibodies consistently yield successful results in every detection attempt for broader use. Our findings indicate that the tested antibody cocktails remained stable over time, which suggests potential for commercialization of this modified Western blot protocol with a wide scope towards multiplex diagnostic application.

Prognostic Value of Immunoglobulin G (IgG) Patterns by Western Blotting Immunodetection in Treated Dogs Previously Infected with Leishmania infantum

Leishmaniasis is a heterogeneous group of neglected tropical diseases with various clinical syndromes, which is caused by obligate intracellular protozoa of the genus Leishmania and transmitted by the bite of a female phlebotomine sandfly. Humans and several animal species are considered as reservoirs of the disease. Among other animal species, dogs are the most important reservoirs in a domestic environment, maintaining the endemic focus of the parasite. The behavior of the disease progression and the clinical symptoms of the disease in the infected dog is mainly associated with depressed cellular immunity and strong humoral response. This study aimed to assess the role of Western blotting in the analysis of the idiotype expression of the two main immunoglobulins (IgG1 and IgG2) in dogs that are naturally infected with Leishmania infantum (L. infantum) and treated with N-methyl meglumine antimoniate. Interestingly, for the first time, our study identified several L. infantum antigen polypeptides (14, 31, 33, 49, 64, 66, 99, and 169 kDa) that more frequently stimulate an immune reaction in recovered dogs after treatment, whereas in the non-recovered group of dogs, four antigen polypeptides of L. infantum with molecular weights of 31, 49, 66, and 115 kDa with unfavorable prognosis were identified. Clearly, these interesting findings confirm the strong association between the detected immunodominant bands and the successful recovery in treated dogs that can be used for differentiating the treated dogs from the untreated dogs, as well as the markers of a favorable or unfavorable prognosis and, as a consequence, the prediction of the clinical outcome of the disease. Likewise, these data could be helpful in the implementation of novel vaccines from the detected antigens.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

Pollutant exposure at wastewater treatment works affects the detoxification organs of an urban adapter, the Banana Bat

The Banana Bat, Neoromicia nana, exploits pollution-tolerant chironomids at wastewater treatment works (WWTWs). We investigated how pollutant exposure impacts the detoxification organs, namely the liver and kidney of N. nana. (i) We performed SEM-EDS to quantify metal content and mineral nutrients, and found significant differences in essential metal (Fe and Zn) content in the liver, and significant differences in Cu and one mineral nutrient (K) in the kidneys. (ii) We performed histological analysis and found more histopathological lesions in detoxification organs of WWTW bats. (iii) We calculated hepatosomatic/renalsomatic indices (HSI/RSI) to investigate whole organ effects, and found significant increases in organ size at WWTWs. (iv) We quantified metallothionein 1E (MT1E), using Western Blot immunodetection. Contrary to predictions, we found no significant upregulation of MT1E in bats at WWTWs. Ultimately, N. nana exploiting WWTWs may suffer chronic health problems from sub-lethal damage to organs responsible for detoxifying pollutants.

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

Distribution and relative activity of matrix metalloproteinase‐2 in human coronal dentin

The presence of matrix metalloproteinase-2 (MMP-2) in dentin has been reported, but its distribution and activity level in mature human coronal dentin are not well understood. The purpose of this study was to determine the MMP-2 distribution and relative activity in demineralized dentin. Crowns of twenty eight human molars were sectioned into inner (ID), middle (MD), and outer dentin (OD) regions and demineralized. MMP-2 was extracted with 0.33 mol·L−1 EDTA/2 mol·L−1 guanidine-HCl, pH 7.4, and MMP-2 concentration was estimated with enzyme-linked immunoabsorbant assay (ELISA). Further characterization was accomplished by Western blotting analysis and gelatin zymography. The mean concentrations of MMP-2 per mg dentin protein in the dentin regions were significantly different (P=0.043): 0.9 ng (ID), 0.4 ng (MD), and 2.2 ng (OD), respectively. The pattern of MMP-2 concentration was OD>ID>MD. Western blotting analysis detected ∼66 and ∼72 kDa immunopositive proteins corresponding to pro- and mature MMP-2, respectively, in the ID and MD, and a ∼66 kDa protein in the OD. Gelatinolytic activity consistent with MMP-2 was detected in all regions. Interestingly, the pattern of levels of Western blot immunodetection and gelatinolytic activity was MD>ID>OD. The concentration of MMP-2 in human coronal dentin was highest in the region of dentin that contains the dentinoenamel junction and least in the middle region of dentin. However, levels of Western blot immunodetection and gelatinolytic activity did not correlate with the estimated regional concentrations of MMP-2, potentially indicating region specific protein interactions.

Anticancer effects of a specific mixture of nutrients in the multidrug-resistant human uterine sarcoma MES-SA/Dx5 and the drug-sensitive MES-SA cell lines

A specific nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has demonstrated a broad spectrum of antitumor activity against a number of cancer cell lines. In this study, our main objective was to investigate the comparative effects of NM on anticancer parameters, such as cytotoxicity, matrix metalloproteinase (MMP) secretion and Matrigel invasion in the human uterine sarcoma drug-resistant MES-SA/Dx5 and the drug-sensitive MES-SA cell lines. In addition we studied the effects of NM on P-glycoprotein (Pgp) on these cell lines. Cell proliferation was evaluated by MTT assay, MMPs by gelatinase zymography, invasion through Matrigel, morphology by H&E and Pgp expression by Western blot analysis and immunodetection using FITC-conjugated antibody and rhodamine123 (Rh123) accumulation and efflux assays. NM exhibited antiproliferative effects on MES-SA/Dx5, by 20% at 50 and 100µg/ml and by 36, 40 and 48% at 250, 500 and 1,000µg/ml, respectively. By contrast, NM treatment of MES-SA cells resulted in significantly increased cytotoxicity: 40, 46, 65 and 72% at 50, 100, 500 and 1,000µg/ml, respectively. In both cell lines, zymography demonstrated a band corresponding to MMP-2 in normal cells and MMP-9 with phorbol 12-myristate 13-acetate treatment. The two MMPs showed dose-response inhibition by NM. As shown by Western blot analysis and immunodetection, NM treatment resulted in a dose-dependent decrease in Pgp expression in the MES-SA/Dx5 cell line. The MES-SA cell line does not exhibit Pgp. NM enhanced the accumulation and efflux of the Pgp substrate, Rh123, in the MES-SA/Dx5 uterine sarcoma cell line but not in the drug-sensitive cell line, MES-SA. Therefore, it can be concluded that NM demonstrates potent anticancer effects in both the drug-resistant and sensitive cell lines and modulates Pgp, suggesting its potential therapeutic effects in drug-resistant as well as sensitive cancers.

TIR8 receptor expression in bovine tissues

The TIR8 receptor (also called SIGIRR) is an orphan member of the TIR superfamily. Its function is still elusive, but it is believed to trigger a negative pathway of regulation of the Toll-like/IL-1 receptor system, crucial for modulating inflammation in gastrointestinal (GI) tract and in other tissues (lung and kidney). The expression pattern of TIR8 in bovine tissues is unknown. Given the importance of GI diseases in cattle, the aim of this investigation was to study the distribution of TIR8 in a wide panel of non-pathologic tissues and organs. TIR8 expression was assessed by Northern blot analysis and further confirmed and comparatively quantified by qualitative and quantitative (Real-Time) PCR. The possible presence of tissue-specific isoforms was determined by Western blot immunodetection, using an anti-human TIR8 polyclonal antibody previously validated in bovine tissues. Similarly to humans and mice, bovine TIR8 was found in the GI tract and kidney. Expression of TIR8 mRNA was also detected in lymph nodes, thymus and thyroid gland. Interestingly, several isoforms of bovine TIR8 were detected in the same organs, suggesting the occurrence of different post-translational processings.

Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.

Induction of the multixenobiotic/multidrug resistance system in various cell lines in response to perfluorinated carboxylic acids.

The multixenobiotic resistance (closely related to multidrug resistance) system controls transport across the plasma membrane as a defense against toxic molecules. Multixenobiotic resistance system consists of an efflux pump, ABCB1 (also named P-glycoprotein, P-gp), and/or a molecule of the ABCC family (also named multiple resistance associated protein, MRP). ABCB1 is able to increase efflux of many low-molecular foreign molecules. Measuring system induction may be used as a biomarker of cell/organism exposure to foreign substances. Various established cell lines were tested for constitutive and induced multixenobiotic resistance proteins by Western blotting immunodetection. The pumping function was indirectly assayed with Rhodamine B by visualization of cell fluorescence in the presence of verapamil. Changes in ABC proteins were measured by flow cytometry after exposition to various perfluorinated carboxylic acids. MCF7 and HeLa cells were found to contain the highest constitutive level of both ABCB1 and ABCC1. HEK293 exhibited much less ABCB1 and no activity of pumping out Rhodamine B. The pumping activity was found to be related to the amount of the cell-type specific 170 kDa ABCB1 protein. An 8-day exposure to 10(-4) M perfluorononanoic acid resulted in about 2-2.5-fold increase of ABCB1 level. That was confirmed also for short times by flow cytometry of cells exposed to perfluorinated acids and its natural congeners. Both ABCB1- and ABCC1-related fluorescence increased along with the carbon chain in acids from C(6) up to C(9) and decreased for C(10). Measuring of multixenobiotic resistance changes in vitro induced by chemicals may be a convenient test for screening for their potential toxicity.

Disseminated Encephalitozoonosis in Captive, Juvenile, Cotton-top (Saguinus oedipus) and Neonatal Emperor (Saguinus imperator) Tamarins in North America

Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.

Effects of ageing on carbonyl stress and antioxidant defense in RBCs of obese Type 2 diabetic patients

In this study we investigated the effects of ageing on the carbonyl stress (protein carbonyls and 4-hydroxy-2-nonenal groups) and glutathione antioxidant defense in red blood cells (RBCs) of obese Type 2 diabetic patients with/without hypertensive complications. To this purpose the following methods were used: spectrophotometry (protein carbonyls, glutathione and glutathione peroxidase assays), immunofluorescence (4-hydroxy-2-nonenal localization), western blotting (immunodetection of carbonylated proteins). The results showed that compared to RBCs of healthy subjects, in obese Type 2 diabetics, ageing is associated with: (i) an increase in the concentration and expression of carbonylated proteins, a marker of oxidative stress; (ii) a decrease of both non-enzymatic and enzymatic endogenous glutathione defenses; (iii) a severely disturbed oxidant/antioxidant balance when obesity was associated with hypertension. The simultaneous insults of high blood pressure, obesity, and diabetes conducted to the highest carbonyl stress, exposure of 4-hydroxy-2-nonenal Michel adducts at the outer leaflet of RBCs plasmalemma, and the lowest glutathione antioxidant potential, particularly in elderly patients. These results can explain the gradual age-dependent diminishment of the detoxification potential of RBCs that at the old age can not overcome the deleterious effects of the high systemic oxidative stress.

New urinary EPO drug testing method using two-dimensional gel electrophoresis

We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. Results The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine samples as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. Conclusion The combination of sample preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.

Binding and Degradation of Heterodimeric Substrates by ClpAP and ClpXP

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

The extracellular aggregation of amyloid beta (Abeta) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Abeta peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Abeta(1-42)-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.

Expression of heat shock protein 27 in human renal cell carcinoma.

Heat shock protein 27 (HSP27, Swiss-Prot accession number P04792) is a component of the large and heterogeneous group of chaperone proteins, and its main functions are inhibition of apoptosis and prevention of aggregation of actin intermediate filament. Modified expression of HSP27 has been described in several cancers including testis, breast, and ovaric cancer. In the present work, 18 renal cell carcinoma (RCC) tissues and homologous normal kidney tissues have been investigated for HSP27 expression by combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) separation and Western blotting immunodetection. The results showed significant differences either in expression and in HSP27 isoform numbers in RCC compared to normal kidney. The average number of isoforms was 21 in RCC and 15 in normal tissues with 4.5-5.9 pI range and 18-29 kDa M(r) range. The overexpression was also observed by immunohistochemistry on tissue sections. Only two of RCC samples showed less isoforms than homologous normal samples. Two isoforms were not detected using anti-Ser82 phosphorylated HSP27 antibody, neither in normal nor in RCC samples. Five of all the immunodetected isoforms were confirmed by mass spectrometry as HSP27, but no evidence of post-translational modifications was pointed out. The numerous isoforms observed in RCC are not consistent with data reported in the literature so far, and they might be due to different post-translational modifications such as phosphorylation and S-thiolation. Since activation of HSP27 seems to be involved in tumor proliferation and drug resistance, it would be crucial to correlate the severity of disease with the different isoforms from RCC samples to generate diagnostic and prognostic markers.

The induction of cyclic nucleotide phosphodiesterase 4 gene (PDE4D) impairs memory in a water maze task

In this study, the effects on memory of intraperitoneal post-training administration of cyclic nucleotide phosphodiesterase (PDE) inhibitors, DC-TA 46 and rolipram, were tested using a visible/hidden-platform water maze task. The effects of these compounds on cyclic nucleotide levels in the hippocampal formation (HF) and striatum (CP) were also assessed, by enzymatic immunoassay (EIA). The results obtained from rats trained in the visible-platform task were not significantly different from controls. On the contrary, the animals trained in the hidden-platform water maze task showed a memory impairment, when injected with DC-TA 46 at maximal dose of 20 mg/kg and with rolipram at 3 and 30 mg/kg doses. The effects of these drugs on cyclic nucleotide levels in HF and CP were observed at 30 min and at 24 h after drug administration. Thirty minutes after drug injection, we observed an increase of cAMP level, both in HF and in CP. Twenty-four hours after the retention test, we observed that in CP the cAMP intracellular level remained high, while in the HF at effective doses both inhibitors induced cAMP PDE activity, determining a decrease of cyclic nucleotide. Semi-quantitative RT–PCR analysis, together with Western blot immunodetection, showed a mRNA and protein induction of PDE4D PDE isoforms, that may account for the increase of PDE activity observed. Our data suggest that, despite cyclic nucleotide increase at 30 min, the fundamental event causing memory impairment, came from the subsequent long time decrease of cAMP levels, due to the post-translational PDE4D induction.

Blood-Brain Barrier Damage Induces Release of α2-Macroglobulin

Blood-brain barrier (BBB) failure occurs in many neurological diseases and is caused in part by activation of proinflammatory factors including matrix metalloproteinases. Counterbalancing, "BBB protective" cascades have recently been described, including NO-mediated interleukin 6 release by glia. Interleukin 6 has been shown to trigger production of matrix metalloproteinase inhibitors such as alpha2-macroglobulin (alpha2M). We hypothesized that BBB failure may result in increased alpha(2)M release by perivascular astrocytes. This was initially tested in patients undergoing iatrogenic BBB disruption by hyperosmotic mannitol for intra-arterial chemotherapy of brain tumors. Serum samples revealed significantly increased levels of alpha2M at 4 h after BBB disruption by hyperosmotic mannitol. In parallel in vitro experiments, we observed a similar increase of alpha2M release by astrocytes under conditions mimicking BBB failure and perivascular edema. For both experiments, protein analysis was initially performed by bidimensional gel electrophoresis and mass spectrometry followed by Western blotting immunodetection. We conclude that, in addition to proinflammatory changes, BBB failure may also trigger protective release of alpha2M by perivascular astrocytes as well as peripheral source.

Isolation of proteins comprising native gene-specific messenger ribonucleoprotein particles using paramagnetic beads

A method for the rapid isolation of proteins comprising native messenger ribonucleoprotein particles (mRNPs), that utilises streptavidin-coated paramagnetic beads, coupled with biotinylated oligodeoxyribonucleotides, to pull down general and transcript-specific mRNP complexes from crude cell-free extracts, is described. Two biotinylated oligo probes were tested for their efficacy in isolating mRNPs from tobacco pollen extracts; a 25-mer oligo(dT) probe for the isolation of total mRNPs, and a transcript-specific oligo complementary to the pollen-specific ntp303 mRNA sequence. Following annealing to the target mRNAs, the oligo probes, with associated ribonucleoprotein particles, were captured by streptavidin-coated paramagnetic particles and pelleted in a magnetic field. After several washing steps, the bound RNPs were released and analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot immunodetection. This one-step mRNP enrichment method could be used either to enhance the detection of low-abundance mRNA-binding proteins in subsequent northwestern and RNA UV cross-linking experiments or the direct identification and biochemical characterisation of RNA-binding proteins.

Heterotrimers Formed by Tumor Necrosis Factors of Different Species or Muteins

Incubation of murine tumor necrosis factor (mTNF) at subnanomolar concentrations results in partial dissociation of the trimers, coinciding with a decrease in bioactivity. Using size-exclusion chromatography, we observed that the conversion of labeled mTNF to monomers is not only prevented by coincubation with an excess of unlabeled mTNF but also with unlabeled human TNF (hTNF). Moreover, after coincubation of mTNF and hTNF four different TNF complexes were revealed by native polyacrylamide gel electrophoresis, viz. homotrimeric mTNF and hTNF, as well as two complexes with an intermediate migration pattern. Analytical gel filtration in combination with native polyacrylamide gel electrophoresis and Western blot immunodetection indicated that these new complexes consisted of heterotrimeric TNF molecules. We conclude that an exchange of monomers takes place during coincubation of two different species of TNF, which results in homotrimeric and heterotrimeric TNF. To assess receptor interaction in vitro, TNF heterotrimeric molecules were used as obtained after incubation of mTNF with labeled hTNF (which only binds to mTNF receptor I) or with labeled mutein mTNF75 (specific for mTNF receptor II). These heterotrimers were retained by both mTNF receptors, which means that the mTNF subunits incorporated in heterotrimeric complexes still can bind to both types of TNF receptor. In addition, the gradual decrease in mTNF bioactivity during preincubation at subnanomolar concentrations was prevented by the presence of mutein mTNF75, which is inactive in an L929 cytotoxicity assay, indicating that heterotrimerization can influence the overall bioactivity.