scholarly journals Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus

1999 ◽  
Vol 343 (2) ◽  
pp. 355-360 ◽  
Author(s):  
Chenhui TANG ◽  
Weiqun LU ◽  
Geoffrey WAINWRIGHT ◽  
Simon G. WEBSTER ◽  
Huw H. REES ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Southern Blotting ◽  
Negative Control ◽  
Sinus Gland ◽  
Methyl Farnesoate ◽  
Putative Signal Peptide ◽  
Cancer Pagurus ◽  
Rapid Amplification ◽  
Regulation Of Growth ◽  
Growth And Reproduction

Methyl farnesoate, the crustacean juvenoid, is synthesized and secreted from the mandibular organs of crustaceans under the negative control of the sinus gland-derived mandibular organ-inhibiting hormone (MO-IH). Previously we isolated and sequenced two isoforms, MO-IH-1 and MO-IH-2, differing by just one amino acid, from sinus glands of the edible crab, Cancer pagurus. We now report the isolation of cDNAs encoding MO-IH-1 and MO-IH-2 by a combination of reverse-transcriptase-mediated PCR in conjunction with 5′ and 3′ rapid amplification of cDNA ends (‘RACE’). Full-length clones of MO-IH-1 and MO-IH-2 encoded a 34-residue putative signal peptide and the mature 78-residue MO-IH sequences. Northern blot analysis of various tissues showed that MO-IH expression is confined to the X-organ (a cluster of perikarya within the eye). Southern blot analysis indicated that there are approx. 10 copies of the gene for MO-IH in C. pagurus. Additional Southern blotting experiments detected MO-IH-hybridizing bands in another Cancer species, C. antennarius. In support of this, an HPLC-radioimmunoassay analysis of sinus gland extracts of C. antennarius and C. magister also revealed MO-IH-like immunoreactivity.

scholarly journals Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection

1998 ◽  
Vol 66 (12) ◽  
pp. 5915-5920 ◽  
Author(s):  
Svena L. McGill ◽  
Russell L. Regnery ◽  
Kevin L. Karem
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Bartonella Henselae ◽  
Negative Control ◽  
Cross Reactivity ◽  
Antibody Activity ◽  
Igg Subclass ◽  
Banding Patterns ◽  
Bartonella Quintana

ABSTRACT Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera againstRickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis,Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintanaantigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG1 and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly toBartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG1. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection withB. henselae or B. quintana.

Retroviral Insertional Mutagenesis Screen in a C/EBPalpha Proliferative Genetic Background.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4342-4342
Author(s):  
Marie S. Hasemann ◽  
Annette B. Sørensen ◽  
Finn S. Pedersen ◽  
Claus Nerlov ◽  
Bo Porse
Keyword(s):  
Myeloid Leukemia ◽  
Insertional Mutagenesis ◽  
Southern Blotting ◽  
Insertion Mutagenesis ◽  
Neutrophil Granulocytes ◽  
Mutagenesis Screen ◽  
T Cell Lymphomas ◽  
Enhancer Binding Protein ◽  
Regulation Of Growth ◽  
Insertional Mutagenesis Screen

Abstract The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours after birth. In contrast, homozygous knockin mice in which the wild type Cebpa allele has been replaced with a mutant allele (BRM2) deficient in repressing the activity of E2F family members, are viable. At 8 weeks of age these animals display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older BRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia. These findings strongly suggest that secondary mutations in other loci must occur during the phenotypic progression. In order to identify genes that cooperate with C/EBPalpha in the development of leukemia in BRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn BRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus and when disease is evident the mice are euthanized and analyzed. As expected the BRM2/BRM2 mice have a shorter latency than wildtype mice (182 vs. 260 days). The mice have enlarged spleen, thymus, and lymph nodes and were further characterized by histology, flow cytometry and Southern blotting in order to determine the hematopoietic phenotypes. Most abundantly was the AML-like phenotype, but also T-cell lymphomas are developing. Finally, the loci carrying retroviral insertions loci are identified through a splinkerette-aided PCR strategy. This study provides a better understanding of the genes involved in the development of myeloid leukemia.

Molecular cloning and characterization of an endo-β-mannanase gene expressed in the lettuce endosperm following radicle emergence

2004 ◽  
Vol 14 (3) ◽  
pp. 267-276 ◽  
Author(s):  
Aoxue Wang ◽  
Jieran Li ◽  
J. Derek Bewley
Keyword(s):  
Blot Analysis ◽  
Cdna Sequence ◽  
Tomato Fruit ◽  
Predicted Amino Acid Sequence ◽  
Degenerate Primers ◽  
High Identity ◽  
Lettuce Seeds ◽  
Radicle Emergence ◽  
Rapid Amplification

Endo-β-mannanase (EC 3.2.1.78), an enzyme that mobilizes the endosperm cell walls of the lettuce (Lactuca sativa L.) seed, increases in activity in the micropylar and lateral regions of this tissue following the completion of germination. Its complementary DNA (cDNA) sequence (LsMan1) was determined using the polymerase chain reaction (PCR) with degenerate primers. The 3‘-end of the cDNA sequence was obtained by 3‘-end rapid amplification of cDNA ends (RACE), and the 5‘-end sequence by genome walking and 5‘-end RACE. The predicted amino acid sequence from the cDNA has a high identity with endo-β-mannanases present in other species (e.g. 67% identity with coffee β-1,4-mannan endohydrolase, 62% identity with tomato fruit endo-β-mannanase). Southern blot analysis suggests the presence of several members of an endo-β-mannanase gene family in the lettuce genome. Several isoforms of the enzyme, including three major ones, were detected by isoelectric focusing. Based on Northern blot analysis, accumulation of the endo-β-mannanase mRNA occurred only after lettuce seeds had germinated, and increased thereafter, although enzyme activity persisted after transcription declined.

scholarly journals Measurement of crustacean hyperglycaemic hormone levels in the edible crab Cancer pagurus during emersion stress

1996 ◽  
Vol 199 (7) ◽  
pp. 1579-1585 ◽  
Author(s):  
S Webster
Keyword(s):  
Sinus Gland ◽  
Short Term ◽  
Air Exposure ◽  
Hormone Levels ◽  
Cancer Pagurus

The effects of emersion stress upon circulating hyperglycaemic hormone (CHH) levels in the edible crab Cancer pagurus were investigated using a highly specific and sensitive radioimmunoassay, with an antiserum directed against HPLC-purified C. pagurus CHH. Emersion resulted in hyperglycaemia and immediate hypoxia, as shown by rapid hyperlactaemia. CHH levels increased dramatically during the first hour of emersion, from almost undetectable levels to around 17 pmol l-1, thereafter increasing to around 30 pmol l-1 after 4 h of emersion. Short-term air exposure experiments demonstrated that significant increases in CHH levels (up to 3.5 pmol l-1) could be detected during the first 15 min of emersion. Although CHH appears to be fairly stable in haemolymph in vitro, injected CHH was cleared extremely rapidly from the haemolymph in vivo. The results suggest that emersion results in rapid, massive and prolonged exocytosis of CHH from the sinus gland. The sensitivity of the assay and the utility of this crab model may be useful in further studies to elucidate the control of CHH release in crustaceans.

scholarly journals Identification of σB-Dependent Genes in Bacillus cereus by Proteome and In Vitro Transcription Analysis

2004 ◽  
Vol 186 (13) ◽  
pp. 4100-4109 ◽  
Author(s):  
Willem van Schaik ◽  
Marcel H. Zwietering ◽  
Willem M. de Vos ◽  
Tjakko Abee
Keyword(s):  
Bacillus Cereus ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Negative Control ◽  
Two Dimensional Gel Electrophoresis ◽  
Transcription Analysis

ABSTRACT The alternative sigma factor σB of the food pathogen Bacillus cereus is activated upon stress exposure and plays a role in the adaptive response of vegetative cells. This study describes the identification of σB-dependent genes in B. cereus. Two-dimensional gel electrophoresis was performed with protein extracts from a σB-overproducing B. cereus strain. Nine protein spots, which were absent from the negative control, were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry or N-terminal sequencing. The σB-dependent expression of the corresponding genes was confirmed by Northern blot analysis with RNA isolated from B. cereus ATCC 14579 and its sigB null mutant. Northern blot analysis also revealed that six other genes were part of σB-dependent operons. The proteins that are predicted to be encoded by the σB-dependent genes include an intracellular protease, a Mg2+ transporter, and a thiamine biosynthesis protein (ThiG). Highly conserved promoter sites were found to precede all σB-dependent genes, with the exception of thiG. By searching the B. cereus genome for this conserved promoter sequence, five more candidate σB-dependent genes were identified. Northern blot analysis and in vitro transcription experiments with a reconstituted B. cereus σB-RNA polymerase holoenzyme confirmed the σB dependency of two of these genes and strongly suggested that two other genes, encoding an oligopeptide-binding OppA-like protein and subunit II of the cytochrome d ubiquinol oxidase, are also σB dependent. In conclusion, σB of B. cereus not only regulates genes directly involved in the stress response but may also control specific metabolic rearrangements.

scholarly journals The Gillenia trifoliata genome reveals dynamics correlated with growth and reproduction in Rosaceae

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Hilary S. Ireland ◽  
Chen Wu ◽  
Cecilia H. Deng ◽  
Elena Hilario ◽  
Ali Saei ◽  
...  
Keyword(s):  
Phenotypic Diversity ◽  
Negative Control ◽  
Fleshy Fruit ◽  
Ancestral Genome ◽  
Important Species ◽  
Syntenic Conservation ◽  
Rosaceae Family ◽  
Improved Model ◽  
Dry Fruit ◽  
Growth And Reproduction

AbstractThe Rosaceae family has striking phenotypic diversity and high syntenic conservation. Gillenia trifoliata is sister species to the Maleae tribe of apple and ~1000 other species. Gillenia has many putative ancestral features, such as herb/sub-shrub habit, dry fruit-bearing and nine base chromosomes. This coalescence of ancestral characters in a phylogenetically important species, positions Gillenia as a ‘rosetta stone’ for translational science within Rosaceae. We present genomic and phenological resources to facilitate the use of Gillenia for this purpose. The Gillenia genome is the first fully annotated chromosome-level assembly with an ancestral genome complement (x = 9), and with it we developed an improved model of the Rosaceae ancestral genome. MADS and NAC gene family analyses revealed genome dynamics correlated with growth and reproduction and we demonstrate how Gillenia can be a negative control for studying fleshy fruit development in Rosaceae.

scholarly journals Aquaporin-1 Protein Levels Elevated in Fresh Urine of Renal Cell Carcinoma Patients: Potential Use for Screening and Classification of Incidental Renal Lesions

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Shilpa Sreedharan ◽  
John A. Petros ◽  
Viraj A. Master ◽  
Kenneth Ogan ◽  
John G. Pattaras ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Blot Analysis ◽  
Renal Cell ◽  
Western Blot Analysis ◽  
Negative Control ◽  
Aquaporin 1 ◽  
Protein Levels ◽  
Fresh Urine

Introduction and Objectives. There are over 65,000 new cases of renal cell carcinoma (RCC) each year, yet there is no effective clinical screening test for RCC. A single report claimed no overlap between urine levels of aquaporin-1 (AQP1) in patients with and without RCC (Mayo Clin Proc. 85:413, 2010). Here, we used archived and fresh RCC patient urine to validate this report.Methods. Archived RCC, fresh prenephrectomy RCC, and non-RCC negative control urines were processed for Western blot analysis. Urinary creatinine concentrations were quantified by the Jaffe reaction (Nephron 16:31, 1976). Precipitated protein was dissolved in 1x SDS for a final concentration of 2 μg/µL creatinine.Results. Negative control and archived RCC patient urine failed to show any AQP1 protein by Western blot analysis. Fresh RCC patient urine is robustly positive for AQP1. There was no signal overlap between fresh RCC and negative control, making differentiation straightforward.Conclusions. Our data confirms that fresh urine of patients with RCC contains easily detectable AQP1 protein. However, archival specimens showed an absence of detectable AQP1 indistinguishable from negative control. These findings suggest that a clinically applicable diagnostic test for AQP1 in fresh urine may be useful for detecting RCC.

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