Intergeneric distribution and immunolocalization of a putative odorant-binding protein in true bugs (Hemiptera, Heteroptera).

1998 ◽  
Vol 201 (1) ◽  
pp. 33-41
Author(s):  
J C Dickens ◽  
F E Callahan ◽  
W P Wergin ◽  
C A Murphy ◽  
R G Vogt
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cellular Localization ◽  
Polyclonal Antiserum ◽  
Adult Males ◽  
Tarnished Plant Bug ◽  
Olfactory Sensilla ◽  
Dense Granules ◽  
Odorant Binding

Lygus antennal protein (LAP) is an olfactory-related protein of the tarnished plant bug Lygus lineolaris (Hemiptera, Heteroptera: Miridae), a hemimetabolous insect. In previous work, a polyclonal antiserum was generated against the N-terminal sequence of LAP; LAP immunoreactivity was strongest in antennae of adult males, but was also present in antennae of adult females and of nymphs. In the current study, LAP immunoreactivity was examined to determine the species specificity and the tissue and cellular localization of LAP expression. Western blot analysis indicated that LAP immunoreactivity was present in the antennae of the male congeners L. lineolaris and L. hesperous, but was not detectable in male antennae of the more distant relatives Podisus maculiventris or Nezara viridula (Hemiptera, Heteroptera: Pentatomidae). Western blot analysis further confirmed that LAP expression was restricted to antennal tissue. Histological analyses showed that LAP expression within the antennae was specifically associated with chemosensory sensilla on the antenna. Within the sensilla, LAP immunoreactivity was distributed throughout the extracellular lumen and was concentrated in dense granules within the cytoplasm of sensillar support cells. LAP immunoreactivity was restricted to a subset of antennal chemosensory sensilla, specifically the multiporous olfactory sensilla. These findings suggest that LAP has an important olfactory function in Lygus sp., possibly related to that of odorant-binding proteins (OBP) found in other insect orders. If so, LAP would be the first OBP-like protein characterized outside the Endopterygota.

scholarly journals Postnatal development and testosterone dependence of a rat epididymal protein identified by neonatal tolerization

Reproduction ◽  
2003 ◽  
pp. 495-507 ◽  
Author(s):  
SA Joshi ◽  
S Shaikh ◽  
S Ranpura ◽  
VV Khole
Keyword(s):  
Western Blot ◽  
Postnatal Development ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antiserum ◽  
Cognate Gene ◽  
Specific Proteins ◽  
Androgen Regulation ◽  
Dose Dependent

A rat epididymal protein of 27 kDa was identified using neonatal tolerization. This study reports the production and characterization of a polyclonal antiserum to this protein. ELISA was used to demonstrate that this antiserum reacts strongly with epididymal sperm proteins, but has little or no reactivity with testicular proteins. Western blot analysis revealed that this polyclonal antiserum recognized a 27 kDa protein extracted from the corpus epididymidis as well as from spermatozoa from the corpus and cauda epididymides, and immunostaining revealed the presence of the protein in the corpus to cauda epididymides. Stronger reactivity was observed in the supranuclear region and stereocilla of principal cells of the corpus epididymidis and in the luminal content of the corpus and cauda epididymides. The testicular section showed no reactivity. Treatment with the antiserum resulted in time- and dose-dependent agglutination of rat spermatozoa. By indirect immunofluorescence, the antiserum localized proteins in the mid-piece region of rat spermatozoa. Studies were carried out to determine the age at which the protein first became apparent during postnatal development. The protein was expressed from day 40 onwards, as demonstrated by western blot analysis. The androgen regulation of this protein was ascertained by castration and supplementation studies. Expression of this protein showed a decline starting at day 14 after castration and by day 21 the protein was absent; however, androgen replacement resulted in the reappearance of the protein. The results of these studies indicate that the protein identified is specific to the epididymis, and is regulated by development and androgens. The importance of epididymis-specific proteins that are regulated by androgens in sperm maturation is discussed, and the need to ascertain the sequence of the protein and clone the cognate gene is indicated.

Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats

2001 ◽  
Vol 280 (6) ◽  
pp. G1209-G1216 ◽  
Author(s):  
Vijay Shah ◽  
Sheng Cao ◽  
Helen Hendrickson ◽  
Janet Yao ◽  
Zvonimir S. Katusic
Keyword(s):  
Portal Hypertension ◽  
Bile Duct ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cellular Localization ◽  
Bile Duct Ligation ◽  
Immunoperoxidase Staining ◽  
Caveolin 1 ◽  
Duct Ligation

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.

scholarly journals First Report of Barley yellow streak mosaic virus-Infected Barley in Alaska

Plant Disease ◽  
2000 ◽  
Vol 84 (5) ◽  
pp. 595-595 ◽  
Author(s):  
N. L. Robertson ◽  
S. K. Brumfield
Keyword(s):  
Electron Microscopy ◽  
Western Blot ◽  
Mosaic Virus ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Virus Disease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyclonal Antiserum ◽  
Mechanical Transmission

Barley yellow streak mosaic virus (BaYSMV) was first described and reported in Montana and Alberta, Canada, more than 17 years ago (1). Since then, it has been detected in two other locations: Pocatello Valley, ID (3), and across the border in Utah. BaYSMV has now been found in the Alaskan interior. In July 1999, dry-land barley (Hordeum vulgare L.) growing in University of Alaska-Fairbanks experimental plots exhibited symptoms similar to those described for BaYSMV, including parallel chlorotic streaks and leaf banding. Mechanical inoculation of Nicotiana benthamiana with diseased barley sap produced systemic mosaic symptoms. As previously reported for BaYSMV sap-transmission tests (2), parallel inoculations to barley plants yielded no symptoms. Electron microscopy of leaf dips and minipurifications of infected N. benthamiana revealed long filamentous particles that matched the size and shape reported for BaYSMV (1). Ultrathin sections of diseased barley and N. benthamiana leaves displayed characteristic virus particles. BaYSMV was confirmed by immuno-sorbent electron microscopy assays (4) and western blot analysis with polyclonal antiserum. Long filamentous BaYSMV particles appeared only on grids coated with BaYSMV antiserum and exposed to diseased N. benthamiana sap. Total protein extracts from diseased barley tissue and inoculated N. benthamiana, as well as with protein extracted from partially purified preparations, were applied to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis minigel and stained with Coomassie blue. Diseased samples, but not healthy controls, contained a protein of ≈33 kDa that was within the size range of a previously described protein from partially purified BaYSMV particles (2). Western blot analysis with an Immuno-Blot alkaline phosphatase assay system (Bio-Rad Laboratories, Hercules, CA) confirmed that the protein reacted with polyclonal BaYSMV. This is the first serological documentation of a BaYSMV-specific protein and that the ≈33-kDa protein is the main antigen recognized by the BaYSMV polyclonal antiserum. Based on virus particle shape and size, symptomology, mechanical transmission host range, and serology, we conclude that BaYSMV is associated with the barley disease observed. Barley yellow streak mosaic virus disease outbreaks are associated with recurring drought and are accompanied by infestations of the brown wheat mite vector, Petrobia latens Müller (1), so it is not surprising that this report coincides with abnormally dry conditions occurring throughout the 1990s in the interior of Alaska. References: (1) N. L. Robertson and T. W. Carroll. Science 240:1188, 1988. (2) N. L. Robertson and T. W. Carroll. Plant Dis. 75:839, 1991. (3) J. S. Skaf et al. Plant Dis. 76:861, 1992. (4) J. S. Skaf and T. W. Carroll. Plant Dis. 79:1003, 1995.

scholarly journals Mixed Virus Infections of Garlic Determined by a Multivalent Polyclonal Antiserum and Virus Effects on Disease Symptoms

Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Miyuki Takaichi ◽  
Takayuki Nagakubo ◽  
Kenji Oeda
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Severe Disease ◽  
Polyclonal Antiserum ◽  
Onion Yellow Dwarf Virus ◽  
Pcr Analysis ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Disease Symptoms

A garlic virus-specific polyclonal antiserum was developed against a mixture of flexuous rodshaped virus particles isolated from mosaic-diseased garlic plants (15). This antiserum was used in Western blot analysis against tissues from mosaic-diseased garlic plants, at least seven viral coat protein (CP) bands (from 38 to 32 kDa) were identified. Using Western blot analysis with Potyvirus-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) analysis, we concluded that three of the seven bands corresponded to CPs of Leek yellow stripe virus (LYSV) (38 kDa) and two different Onion yellow dwarf virus (OYDV) strains (35.5 or 34 kDa). The 35 kDa band corresponded to the CP of GV1-Carlavirus, and the other four bands, 36, 35 (not GV1), 33, and 32 kDa, were identified as the CPs of four mite-borne viruses, based on RT-PCR analysis. Based on the molecular weights of CP, mixed infections of Potyvirus, Carlavirus, and mite-borne viruses were characterized. LYSV causes apparent disease symptoms in garlic plants, however, little reduction in bulb weights. Conversely, garlic plants infected with three different mite-borne viruses expressed weak symptoms and yield losses. Mixed infections of OYDV, the mite-borne viruses, and LYSV caused severe disease symptoms and considerable reduction of bulb weights.

scholarly journals Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus

2006 ◽  
Vol 96 (6) ◽  
pp. 560-566 ◽  
Author(s):  
M. Turina ◽  
M. Ciuffo ◽  
R. Lenzi ◽  
L. Rostagno ◽  
L. Mela ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Aphid Transmission ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coated Plate ◽  
Ranunculus Asiaticus ◽  
Das Elisa

Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.

scholarly journals Characterization of monoclonal antibodies against ovine prolactin: suitability for use in immunocytochemical analysis of rat prolactin.

1990 ◽  
Vol 38 (1) ◽  
pp. 117-122 ◽  
Author(s):  
J G Scammell ◽  
M G Scott ◽  
K K Outlaw ◽  
M E Thompson ◽  
S J O ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyclonal Antiserum ◽  
Amino Acid Sequences ◽  
Rat Tissues ◽  
Rat Pituitary ◽  
Ovine Prolactin ◽  
Rat Pituitary Tumor Cells

The aim of this study was to identify a monoclonal antibody (MAb) suitable for use in the immunocytochemical localization of prolactin in rat tissues. We took advantage of the conservation of certain amino acid sequences in prolactin among species by examining the crossreactivity patterns of five MAb, originally generated to ovine prolactin, with rat prolactin by enzyme-linked immunoassay (ELISA), Western blot analysis, and immunocytochemistry. Two of five antibodies (17D9 and 6F11) showed reactivity with 100 ng of immobilized rat prolactin (NIH RP-3) by ELISA, 6F11 reacting more strongly than 17D9. Only 6F11 reacted with prolactin in lysates of GH4C1 rat pituitary tumor cells by Western blot analysis. When we examined the crossreactivity of the MAb with rat prolactin in monolayer cultures of GH4C1 cells by indirect immunofluorescence, we found that both 17D9 and 6F11 reacted strongly with the cultures. The distribution of staining with 17D9 or 6F11 was coincident with staining with a polyclonal antiserum to rat prolactin. Preabsorption of the antibodies with a 20-fold excess of purified rat prolactin abolished the staining of GH4C1 cell cultures with either antibody. Therefore, we have selected from a series of MAb raised to ovine prolactin two antibodies (17D9 and 6F11) that react specifically with rat prolactin in immunocytochemical studies, whereas 6F11 also reacts strongly with rat prolactin by ELISA and Western blot analysis.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

Sign in / Sign up

Export Citation Format

Share Document