scholarly journals Culex quinquefasciatus Late Trypsin Biosynthesis Is Translationally Regulated by Trypsin Modulating Oostatic Factor

2021 ◽  
Vol 12 ◽  
Author(s):  
Dov Borovsky ◽  
Peter Verhaert ◽  
Pierre Rougé ◽  
Charles A. Powell ◽  
Arnold De Loof
Keyword(s):  
Culex Quinquefasciatus ◽  
Northern Blot ◽  
Blood Meal ◽  
Blot Analysis ◽  
High Performance ◽  
Northern Blot Analysis ◽  
Three Dimensional ◽  
Transcript Abundance ◽  
Trypsin Activity ◽  
Trypsin Modulating Oostatic Factor

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.

scholarly journals IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Roni M. Shtein ◽  
Susan G. Elner ◽  
Zong-Mei Bian ◽  
Victor M. Elner
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Stromal Cells ◽  
Blot Analysis ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Statistical Significance ◽  
Dependent Manner ◽  
Corneal Inflammation ◽  
Chemotactic Protein

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells.Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student'st-test.Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05) after corneal stromal cell stimulation with LPS.Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

scholarly journals A rapid and accurate method for quantitating total RNA transferred during Northern blot analysis

1988 ◽  
Vol 16 (5) ◽  
pp. 2354-2354 ◽  
Author(s):  
Nathalie Denis ◽  
Daniel Corcos ◽  
Jacques Kruh ◽  
Alain Kitzis
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Accurate Method ◽  
Total Rna

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis

FEBS Letters ◽  
1995 ◽  
Vol 372 (2-3) ◽  
pp. 151-156 ◽  
Author(s):  
Masato Katsuyama ◽  
Nobuhiro Nishigaki ◽  
Yukihiko Sugimoto ◽  
Kimiko Morimoto ◽  
Manabu Negishi ◽  
...  
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Prostaglandin E

A procedure for Northern blot analysis of native RNA

1986 ◽  
Vol 159 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Edouard W. Khandjian ◽  
Claude Méric
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis

The expression of NG2 proteoglycan in the developing rat limb

Development ◽  
1991 ◽  
Vol 111 (4) ◽  
pp. 933-944 ◽  
Author(s):  
A. Nishiyama ◽  
K.J. Dahlin ◽  
W.B. Stallcup
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Core Protein ◽  
Staining Intensity ◽  
Limb Bud ◽  
Protein Size ◽  
Glial Progenitor Cells ◽  
Ng2 Proteoglycan ◽  
Developing Rat

NG2 is a chondroitin sulfate proteoglycan previously found to be expressed by glial progenitor cells of the O2A lineage. We have examined the expression of NG2 in the developing rat limb by immunohistochemistry and northern blot analysis. Staining of embryonic day 14 (E14) rat limb bud sections with polyclonal and monoclonal anti-NG2 antibodies reveals reactivity in the precartilaginous mesenchymal condensation. The staining intensity increases with the differentiation of chondrocytes until E16. NG2 staining is not detected in the mature hypertrophic chondrocytes of E17 and postnatal day 3 (P3) limbs even after treatment of the sections with hyaluronidase or collagenase. Immuno-precipitations with anti-NG2 antibody using 125I-labeled limb cells in culture showed a 400 to 800 × 10(3) Mr proteoglycan species with a core protein size of 300 × 10(3) Mr, comparable to NG2 from O2A cells and neural cell lines. Northern blot analysis reveals the expression of an 8.9 kb mRNA in E16 limbs and at a lower level in P1 cartilage. The northern blot analyses also show that NG2 is distinct from the large aggregating proteoglycan of the cartilage. Our results indicate that in the developing limb cartilage, as in the differentiating oligodendrocytes, NG2 is present on immature cells in the process of differentiating, but its expression is downregulated as terminal differentiation of chondrocytes takes place.

PKC-lambda is the atypical protein kinase C isoform expressed by immature ventricle

1997 ◽  
Vol 272 (4) ◽  
pp. H1636-H1642
Author(s):  
V. O. Rybin ◽  
P. M. Buttrick ◽  
S. F. Steinberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Ventricular Myocardium ◽  
Adult Rat ◽  
Atypical Pkc ◽  
Kinase C ◽  
Pkc Zeta

We recently identified a developmental decline in protein kinase C (PKC) isoform expression, at the level of the protein, in rat ventricular myocardium. To investigate mechanisms regulating PKC isoform expression in cardiac tissue, this study uses Northern blot analysis to compare the abundance of PKC isoform mRNAs in neonatal and adult rat ventricular myocardium. PKC-epsilon protein and mRNA were detected in both neonatal and adult rat ventricular myocardial preparations. In contrast, coordinate postnatal declines in the abundance of PKC-alpha and PKC-delta proteins and transcripts were identified. An antiserum raised against the COOH-terminal sequence of PKC-zeta detected abundant immunoreactivity in neonatal, but not adult, ventricular myocytes. However, PKC-zeta transcripts were not detectable in the heart either by Northern blot analysis or a reverse transcriptase-polymerase chain reaction approach, indicating that neither the myocytes nor the contaminating cellular elements in the heart express PKC-zeta. Rather, PKC-lambda, another atypical PKC isoform that is structurally highly homologous to PKC-zeta, was detected at the protein and mRNA level in neonatal, but not adult, ventricular myocardium. Taken together, these results establish that developmental declines in calcium-sensitive, novel, and atypical PKC isoforms are paralleled by changes in the levels of the mRNAs encoding these proteins, suggesting transcriptional regulation of PKC during normal cardiac development. The results of this study further identify PKC-lambda as the atypical PKC isoform expressed by the immature ventricle.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

2004 ◽  
Vol 16 (8) ◽  
pp. 763 ◽  
Author(s):  
Han-Seung Kang ◽  
Chae-Kwan Lee ◽  
Ju-Ran Kim ◽  
Seong-Jin Yu ◽  
Sung-Goo Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oestrous Cycle ◽  
Mrna Levels ◽  
Rat Uterus ◽  
Ovx Rats

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P4)-injected OVX rats (OVX v. OVX + P4 pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P4 treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [α32P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P4 pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P4 animals. The genes for nuclear factor I–XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin β-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P4 rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P4. One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17β-oestradiol (E2) and P4, although P4 administration upregulated gene expression to a greater extent than injection of E2. These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E2 and P4, which are secreted periodically during the oestrous cycle.

scholarly journals The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 2158-2166 ◽  
Author(s):  
David Vicent ◽  
Eleftheria Maratos-Flier ◽  
C. Ronald Kahn
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Branch Point ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Geranylgeranyl Diphosphate ◽  
Protein Prenylation ◽  
Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

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