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WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.

WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.

English: In this study, sequences encoding VP2 and VP6 rotavirus structural proteins from rotavirus strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were used in construction of wide-range yeast expression vectors containing the ORFs encoding rotavirus structural proteins VP2 and VP6. VP2 and VP6 sequences were codon-optimized for expression in yeast strains K. lactis, A. adeninivorans and Pichia pastoris/Hansenula polymorpha. Wide-range yeast expression vectors containing either VP6 or VP2 yeast optimized ORFs were constructed for expression of single proteins in different yeast strains. Dual vectors containing both VP6 and VP2 yeast optimized ORFs were constructed to allow simultaneous expression of proteins in different yeast strains and to enable the formation of double-layered rotavirus-like particles. A total of eight yeast strains namely: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris Candida deformans and Arxula adeninivorans were selected for screening. Saccharomyces cerevisiae was included as a positive control as triple-layered rotavirus virus-like particles (tlRLPs) have been successfully produced in this yeast before. All nine yeast strains were successfully transformed with pKM177 vectors containing yeast codon-optimized VP6 ORFs. However, only six yeast strains indicated positive VP6 ORF integration. The other three yeast strains indicated no VP6 integration but hygromycin B integration suggesting that the vectors successfully integrated in these yeast strains but VP6 ORF was lost. Integration of both rotavirus VP2/6 ORFs was evident in all yeast strains transformed with the dual expression vectors. A control to monitor rotavirus VP6 protein expression in yeast was successfully prepared in bacterial cells. The positive control indicated specific reaction when polyclonal rotavirus antibody raised against Nebraska calf diarrhoea rotavirus strain. Rotavirus VP6 expression in yea, Afrikaans: In hierdie studie, basispaaropeenvolgings wat kodeer vir VP2 en VP6 rotavirus strukturele proteïene van rotavirus stam RVA/Human-wt/ZAF/GR10924/1999/G9P [6] is gebruik in die konstruksie van ‘n wye verskeidenheid gis uitdrukkingsvektore met rotavirus strukturele proteïene VP2 en VP6 oopleesrame. VP2 en VP6 basispaaropeenvolgings is kodon geoptimaliseer vir uitdrukking in gis stamme K. lactis, A. adeninivorans en P. pastoris/H. polymorpha. Wye verskeidenheid gis uitdrukkingsvektore wat geoptimaliseerde oopleesrame van VP6 of VP2 bevat, is gebou vir uitdrukking van enkel proteïene in verskillende gisstamme. Vektore wat beide VP6 en VP2 geoptimaliseerde oopleesrame bevat is gebou vir gelyktydige uitdrukking van proteïene in verskillende gisstamme en om die vorming van dubbellaag rotavirus-agtige partikels toe te laat. 'n Totaal van agt gisstamme naamlik: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Candida deformans en Arxula adeninivorans is gekies vir sifting. Saccharomyces cerevisiae is ingesluit as 'n positiewe kontrole aangesien trippellaag rotavirus-agtige partikels (tlRLPs) voorheen suksesvol in hierdie gis geproduseer is. Al nege gisstamme is suksesvol getransformeer met pKM177 vektore wat gis geoptimaliseerde kodon VP6 oopleesrame bevat. Daar was egter slegs ses gisstamme waarin positiewe VP6 ORF integrasie gevind is. Die oorblywende drie gisstamme het higromisien B integrasie getoon, wat daarop dui dat die vektore suksesvol geïntegreer het in hierdie gisstamme, maar dat VP6 oopleesraam verloor is. Integrasie van beide rotavirus VP2/6 oopleesrame was suksesvol vir al die gis stamme wat met die dubbele uitdrukkingsvektore getransformeer is. Rotavirus VP6 proteïenuitdrukking is suksesvol uitgevoer in bakteriële selle. Die positiewe kontrole het ʼn spesifieke reaksie getoon met poliklonale rotavirus teenliggaampies wat teen Nebraska calf diarrhoea rotavirus opgewek is, National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF)

Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 hours after treatment but returned to control levels 24 hours after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and Western blot experiments indicates that GNP-treatment up-regulated B-lymphocyte-induced maturation protein 1 (blimp1) and down-regulated paired box 5 (pax5). Immunostaining for Blimp1 and Pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity

The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to antib

Protease-activated receptor 2 (PAR2) is a G proteincoupled receptor activated by intramolecular docking of a tethered ligand that is released by the actions of proteases, mainly of the serine protease family. Here, we evaluate four commercially available anti-PAR2 antibodies, SAM11, C17, N19 and H99, demonstrating marked differences in the ability of these reagents to detect the target receptor in Western blot, immunocytochemical and flow cytometry applications. In Western blot analysis, we evaluated antibody reactivity against both ectopic and endogenous receptors. Against material from transfected cells, we show that SAM11 and N19, and to a lesser extent C17, but not H99, are able to detect ectopic PAR2. Interestingly, these Western blot analyses indicate that N19 and C17 detect conformations of ectopic PAR2 distinct to those recognised by SAM11. Significantly, our data also indicate thatWestern blot signal detected by SAM11 and C17, and much of the signal detected by N19, against cells endogenously expressing PAR2 is non-specific. Despite confounding nonspecific signals, we were able to discern N19 reactivity against endogenous PAR2 as a broad smear that we also observed in ectopically expressing human and mouse cells and that is sensitive to loss of N-glycosylation. In immunocytochemistry analysis, each antibody is able to detect ectopic PAR2 although it appears that H99 detects only a subset of the ectopically expressed receptor. In addition, SAM11 and N19 are able to detect both ectopic and endogenous cell surface PAR2 by flow cytometry. In summary: (1) each antibody can detect ectopic PAR2 by immunocytochemical analysis with SAM11 andN19 suitable for cell surface detection of both ectopic and endogenous receptor by flow cytometry; (2) in Western blot analysis, N19, SAM11 and C17 can detect ectopically expressed PAR2, with only N19 able to detect the endogenous receptor by this technique and (3) in each of these approaches, appropriate controls are essentia

The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813-829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC-mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F-L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity "GNAL" domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to antib

There is abundant evidence to suggest that epigenetic DNA methylation changes may appear early during prostate cancer (PCa) development than genetic changes, and are more common and consistent. The purpose of this study is to investigate whether aberrant DNA methylation occurs as a function of age and if it accompanies neoplastic transformation of the human prostate. RESULTS: Using methylated CpG island amplification coupled with promoter microarray and also genome-wide DNA methylation analysis, I have identified several novel genes that are differentially methylated in normal and PCa tissues. In addition, I have also identified novel methylated patterns among preinvasive and cancerous lesions of the prostate. Quantitative methylation analysis revealed significant differences (p 0.05) in methylation level for several genes in African-American (AA) samples versus Caucasian (Cau) samples. Furthermore, regression analysis revealed significantly higher methylation for NKX2-5 (p = 0.008) and TIMP3 (p = 0.039) genes in normal prostate tissue samples from AA versus Cau, and a statistically significant association of methylation with age for NKX2-5 (p = 0.03) in AA versus Cau. In addition, functional studies demonstrate that NKX2-5 may have tumor suppressor function in PCa cells. CONCLUSION: I have identified several novel methylated genes as potential (ethnic sensitive) biomarkers for early disease detection and for distinguishing indolent from aggressive PCa., The original document contains color images.

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-cR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88-mediated proinflammatory cytokine responses. We also reported that MyD882/2 mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II+ cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-a and IL-1b. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-a and IL-1b. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines., Published in PLoS ONE, v6 n1 p1-12, 20 Jan 2011.

During this second year of the project, we established multiple primary cell strains from normal controls and SSc patients. We performed stimulation assays with silica in 82 primary fibroblast strains. Our results showed that silica activate fibroblasts toward fibrotic changes. However, different fibroblast strains obtained from different individuals showed different responses in terms of the gene expression of the ECM components. Using longitudinal linear models in analysis of association between specific genotypes and dynamic changes of gene expression of the fibroblasts in responding to silica stimulation, we identified that specific SNPs were associated with either single gene expression, or paired gene expression such as CTGF/SPARC and COL1A2/COL3A1, which suggest a strong biological correlation between these genes. Moreover, some SNPs and/or their corresponding genes were found to be associated with both SSc susceptibility and the fibrotic changes of human fibroblast in response to silica stimulation. These SSc susceptibility genes include not only previously identified ones, but also some novel ones, such as HLA-DPB1 and APBA1. These observations supported our original proposal that genetic elements within SSc fibroblasts might contribute to susceptibility to fibrotic process. Integrative studies of genetic and environmental factors with human fibroblasts may facilitate the discovery of potential pathogenesis of SSc. In this year, we will continuously obtain more human fibroblasts, especially SSc fibroblasts. We also will continuously perform stimulation assays of newly obtained human fibroblasts for a better chance to identify genetic components inside SSc patients contributing to susceptibility to environmental stimuli. Meanwhile, we will try to explore which specific bio-pathways associated with which specific genetic factors involved in silica induced fibrotic changes. Therefore, our studies are fulfilled with original proposal in the grant., The original document contains color images.

Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes bacillary dysentery by invading the colonic epithelium. Previous research has shown that Shigella induces a rapid cell death in macrophages. In infected epithelial cells however only a stress response is observed and the eukaryotic cells remain viable during infection. Since S. flexneri utilizes epithelial cells for replication and survival, the hypothesis of this dissertation is that S. flexneri inhibits apoptosis in epithelial cells in order to establish a replicative niche inside the host. An apoptosis assay was developed by adding staurosporine, a chemical inducer of the intrinsic pathway of apoptosis, to the standard invasion assay.

Methanococcoides burtonii is a cold-adapted archaeon isolated from permanently cold (1-2 deg C), methane saturated waters in Ace Lake, Antarctica. M. burtonii is a motile, flagellated microbe that uses methylated carbon compounds for growth (methylotrophy), such as methanol and trimethylamine. Although adapted to the cold, M. burtonii is capable of growth at much higher temperatures, with the highest (= optimal) growth rate occurring at 23 deg C, and a maximum growth temperature of 28 deg C. We have employed two separate but integrated approaches for investigating the basis of cold adaptation in M. burtonii: targeted protein studies, and global proteomic analysis. Our first approach focuses on the study of the chaperonin (Cpn60) complex, which is important for the correct folding of proteins inside the cell. Our second approach aims to determine how protein expression changes in the entire cell at different growth temperatures, using isotope labeling of proteins and liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS). Our research aims to provide fundamental knowledge about the molecular mechanisms of adaptation to growth at cold temperatures in archaea. This will enable us to identify molecular strategies that have evolved in order to cope with extreme cold, with the cell in a state of permanent cold stress. This is interesting in terms of both the limits of archaeal adaptation to low temperature extremes, and in considering whether archaeal mechanisms for cold adaptation may be extended to other systems.

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than M. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode.

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode.

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode.

About biological affecting of flavonoids on animal organisms is known less, thus we selected flavonoids, flavanones and flavones, and their glycosides, which were examined as potential inducers of cytochrome(s) P450 when administrated by gavages into experimental male rats. The study was focused on induction of CYP1A1, the major cytochrome P450 involved in carcinogen activation. The data obtained demonstrate the necessity of taking into account not only ability of flavonoids to bind to Ah receptor (induction factor) but also to concentrate on their distribution and metabolism (including colon microflora) in the body. After that we examined certain flavonoids as potential inducers of cytochrome P450, we wanted to suggest and optimize suitable electrochemical technique for determination of selected flavonoids (quercetin, quercitrin, rutin, chrysin and diosmin) in body liquids. For these purposes, we selected square wave voltannetry using carbon paste electrode. Primarily we aimed on investigation of their basic electrochemical behaviour. After that we have optimized frequency, step potential and supporting electrolyte. Based on the results obtained, we selected the most suitable conditions for determination of the flavonoids as follows: frequency 180 Hz, step potential 1.95 mV/s and phosphate buffer of pH 7 as supporting electrolyte. Detection limits (3 S/N) of the flavonoids were from units to tens of nM except diosmin, where the limit were higher than M. In addition, we attempted to suggest a sensor for analysis of flavonoids in urine. It clearly follows from the results obtained that flavonoids can be analysed in the presence of animal urine, because urine did not influence much the signals of flavonoids (recoveries of the signals were about 90 %).

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery. In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases., Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery. In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases., Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.

This project studied the genetic basis of tumor pathogenesis in neurofibromatosis 1 (NF1). This work tested the two-hit hypothesis in NF1 tumors (neurofibromas and MPNSTs), and involvement of other loci such as TP53. Human tumor samples were collected, with development of cell cultures from some. Discoveries: the two-hit mechanism operates in at least a large proportion of neurofibromas (which may include methylation as a mechanism); there is a genetically abnormal clonal Schwann cell component in these tumors; somatic isodisomy may be present in loss of heterozygosity cases; the TP53 gene (and RB1 and P73) is implicated only in MPNSTs; and the ErbB2 gene may be a modifier of NF1.

As a result of its interaction with a specific receptor, inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular calcium. The metabolism of InsP3 is rather complex: InsP3 3-kinase produces Inositol 1,3,4,5-tetrakisphosphate (InsP4), a putative second messenger also involved in the intraneuronal calcium homeostasis. The distribution of the messenger RNA coding for the recently cloned InsP3 3-kinase was studied in the developing rat brain by using oligonucleotides derived from the rat cDNA sequence and in situ hybridization combined with Northern blot analysis. In addition, the locations of the enzyme were determined by immunohistochemistry in combination with Western blot analysis. By Northern blot and Western blot analyses on rat brain, the kinase was not detected in the embryo, was first found slightly at birth, and reached adult levels around 2-3 postnatal weeks. These findings were confirmed in the different positive regions by in situ hybridization conducted at the macroscopic level. At the cellular level, the mRNA was found exclusively in the neuronal populations previously demonstrated in the adult. The levels of transcripts per neuron were however higher in the adult than in the neonate brain. The enzyme mRNA could be detected first at postnatal day 0, (birth, P0) in the perikarya of the cerebellar Purkinje cells, followed at P4 by the hippocampal CA1 pyramidal cells and granule cells of the dentate gyrus and finally, at P9, by a majority of the neurons in the cortical layers II-III and V, especially in the frontal cortex and cingulate cortex; claustrum; caudate, putamen, accumbens, olfactory tubercle and calleja islets; anterior olfactory nucleus; taenia tecta; piriform piriform cortex; dorsolateral septum; bed nucleus stria terminalis; amygdala; hippocampal CA2-4 sectors and subiculum. By immunohistochemistry, the enzyme was initially found in the periphery of the cell bodies of the neonatal neurons; was progressively enriched in the developing dendrit, Journal Article, Research Support, Non-U.S. Gov't, FLWNA, SCOPUS: ar.j, info:eu-repo/semantics/published

As a result of its interaction with a specific receptor, inositol 1,4,5-trisphosphate (InsP3) mobilizes intracellular calcium. The metabolism of InsP3 is rather complex: InsP3 3-kinase produces Inositol 1,3,4,5-tetrakisphosphate (InsP4), a putative second messenger also involved in the intraneuronal calcium homeostasis. The distribution of the messenger RNA coding for the recently cloned InsP3 3-kinase was studied in the developing rat brain by using oligonucleotides derived from the rat cDNA sequence and in situ hybridization combined with Northern blot analysis. In addition, the locations of the enzyme were determined by immunohistochemistry in combination with Western blot analysis. By Northern blot and Western blot analyses on rat brain, the kinase was not detected in the embryo, was first found slightly at birth, and reached adult levels around 2-3 postnatal weeks. These findings were confirmed in the different positive regions by in situ hybridization conducted at the macroscopic level. At the cellular level, the mRNA was found exclusively in the neuronal populations previously demonstrated in the adult. The levels of transcripts per neuron were however higher in the adult than in the neonate brain. The enzyme mRNA could be detected first at postnatal day 0, (birth, P0) in the perikarya of the cerebellar Purkinje cells, followed at P4 by the hippocampal CA1 pyramidal cells and granule cells of the dentate gyrus and finally, at P9, by a majority of the neurons in the cortical layers II-III and V, especially in the frontal cortex and cingulate cortex; claustrum; caudate, putamen, accumbens, olfactory tubercle and calleja islets; anterior olfactory nucleus; taenia tecta; piriform piriform cortex; dorsolateral septum; bed nucleus stria terminalis; amygdala; hippocampal CA2-4 sectors and subiculum. By immunohistochemistry, the enzyme was initially found in the periphery of the cell bodies of the neonatal neurons; was progressively enriched in the developing dendrit, Journal Article, Research Support, Non-U.S. Gov't, FLWNA, SCOPUS: ar.j, info:eu-repo/semantics/published

Electron micrographs have been obtained showing Thermoplasma acidophilum in amoeboid shapes attached to the surface of sulfur crystals. (2) Anti-actin antibodies reacted weakly with a 90 kDa protein in Western Blot analysis. Other cytoskeleton-related antibodies did not react. (3) Cytoplasmic extracts of T. acidophilum increased in viscosity upon incubation. Optimal conditions were approximately physiological. During incubation a subset of previously-soluble proteins became sedimentable by low speed centrifugation. (4) Extraction with nonionic detergents left an insoluble residue of several protein species. (5) A calcium-affected protein has been identified as ferredoxin. It binds Ca(++) with an affinity near 10 to the 6th power/M. (6) By autoradiography using phosphorus-32, hundreds of proteins are phosphorylated in vivo in T. acidophilum. Keywords: Archaebacteria, Superoxide dismutase, Algae.

B. anthracis Sterne cells were treated with monoclonal antibodies (MAbs) to protective antigen and subjected to the indirect-fluorescent antibody assays. Fluorescence was discernible throughout the surface of cells. Extraction of the cells with 8M urea or 1% SDS brought about a total loss of fluorescence. Pretreatment of the same cells with trypsin seemed to render the fluorescence somewhat stronger, with distinct patterns of granularity. Preincubation of the cells with soy bean agglutinin seemed to bring about a patchiness in the fluorescence distribution. Polysaccharide, Indirect fluorescence antibody assays, Polysaccharide-monoclonals, Immunolabelling-TEM, Western blots, periodate oxidation, Smith degradation. 13C-NMR.

A panel of 11 murine monoclonal antibodies directed against dengue type 2 was evaluated for antigen specificity by dot immunobinding assay and Western blot analysis and for in vitro and in vivo biological activities. Nine of the 11 monoclonal antibodies reacted with viral E-glycoprotein based on the Western blot analysis; one reacted with a 36 Kd protein present in dengue-infected C6/36 mosquito cells. The nine E-glycoprotein-reactive monoclonal antibodies also neutralized dengue 2 virus in a plaque reduction assay. Of the neutralizing monoclonal antibodies, five passivelly protected mice in vivo against lethal intracerebral dengue 2 challenge. The protective monoclonal antibodies were directed against viral determinants that fell into at least three spatialy separate families of epitopes on E-glycoprotein, the antigenicities of which were preserved after heat/detergent denaturation. Reprints.

© 2015 Elsevier Ltd. Neuropeptide Y (NPY) playsa trophic role in the nervous and vascular systems and incardiac hypertrophy. However, there i s no report concerning the expression of NPY and its receptors in the heart during postnatal development. In the current study, immunohistochemistry and Western blot analysis was used to label NPY, and Y1R, Y2R, and Y5R receptors in the heart tissue and intramural cardiac ganglia from rats of different ages (newborn, 10 days old, 20 days old, 30 days old, 60 days old, 1 year old, and 2 years old). The obtained data suggest age-dependent changes of NPY-mediated heart innervation. The density of NPYimmunoreactive (IR) fibers was the least in newborn animals and increased in t h e first 20 days of life. In t he atria of newborn and 10-day-old rats, NPY-IR fibers were more abundant compared with t h e ventricles. The vast majority of NPY-IR fibers also contained tyrosine hydroxylase, a key enzyme in catecholamine synthesis. The expression of Y1R increased between 10 and 20 days oflife. Faint Y2R immunoreactivity was observed in the atria and ventricles of 20-day-old and older rats. In contrast, the highest level of the expression of Y5R was found in newborn pups comparing with more adult rats. All intramural ganglionic neurons were also Y1R-IR and Y5R-IR and Y2R-negative in all studied animals. Thus, the increasing of density of NPY-containing nerve fibers accompanies changes in relation of different subtypes of NPY receptors in the heart during development.

© 2015 Elsevier Ltd. Neuropeptide Y (NPY) playsa trophic role in the nervous and vascular systems and incardiac hypertrophy. However, there i s no report concerning the expression of NPY and its receptors in the heart during postnatal development. In the current study, immunohistochemistry and Western blot analysis was used to label NPY, and Y1R, Y2R, and Y5R receptors in the heart tissue and intramural cardiac ganglia from rats of different ages (newborn, 10 days old, 20 days old, 30 days old, 60 days old, 1 year old, and 2 years old). The obtained data suggest age-dependent changes of NPY-mediated heart innervation. The density of NPYimmunoreactive (IR) fibers was the least in newborn animals and increased in t h e first 20 days of life. In t he atria of newborn and 10-day-old rats, NPY-IR fibers were more abundant compared with t h e ventricles. The vast majority of NPY-IR fibers also contained tyrosine hydroxylase, a key enzyme in catecholamine synthesis. The expression of Y1R increased between 10 and 20 days oflife. Faint Y2R immunoreactivity was observed in the atria and ventricles of 20-day-old and older rats. In contrast, the highest level of the expression of Y5R was found in newborn pups comparing with more adult rats. All intramural ganglionic neurons were also Y1R-IR and Y5R-IR and Y2R-negative in all studied animals. Thus, the increasing of density of NPY-containing nerve fibers accompanies changes in relation of different subtypes of NPY receptors in the heart during development.