scholarly journals Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase-PL-I Fusion Protein

1998 ◽  
Vol 46 (6) ◽  
pp. 737-743 ◽  
Author(s):  
Heiner Müller ◽  
Guoli Dai ◽  
Michael J. Soares
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Colorimetric Assay ◽  
Biological Activities ◽  
Placental Lactogen ◽  
Kidney Cells ◽  
Fetal Kidney ◽  
Transfected Cells ◽  
Tissue Sections ◽  
Labeling System

The rat placenta expresses a family of genes related to prolactin (PRL). Target tissues and physiological roles for many members of the PRL family have yet to be determined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I (PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat PL-I (AP-PL-I). The AP-PL-I construct was stably expressed in 293 human fetal kidney cells, as was the unmodified AP vector that served as a control. AP activity was monitored with a colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and PRL-like biological activities of the AP-PL-I fusion protein were demonstrated by immunoblotting and the Nb2 lymphoma cell proliferation assay, respectively. AP-PL-I specifically bound to tissue sections known to express the PRL receptor, including the ovary, liver, and choroid plexus. Binding of AP-PL-I to tissues was specific and could be competed with ovine PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and suggest that this labeling system may also be useful for monitoring the actions of other members of the PRL family.

Preparation, spectral properties and biological activities of 5-bromo-6-methyl-2'-deoxyuridine and 5-iodo-6-methyl-2'-deoxyuridine

1980 ◽  
Vol 45 (8) ◽  
pp. 2364-2370 ◽  
Author(s):  
Antonín Holý ◽  
Erik De Clercq
Keyword(s):  
Antiviral Activity ◽  
Spectral Properties ◽  
De Novo ◽  
Biological Activities ◽  
Rabbit Kidney ◽  
Kidney Cells ◽  
Cd Spectra ◽  
Methyl Derivatives ◽  
Primary Rabbit

Reaction of 3',5'-di-O-benzoyl-6-methyl-2'-deoxyuridine (IIa) with elementary bromine or iodine afforded 5-halogeno derivatives IIc and IId which on methanolysis gave 5-bromo-6-methyl-2'-deoxyurine (Ic) and 5-iodo-6-methyl-2'-deoxyurine (Id), respectively. The CD spectra of Ic, Id and 6-methyl-2'-deoxyuridine (Ia) are compared and discussed with regard to determination of the nucleoside conformation. Unlike 5-bromo- and 5-iodo-2'-deoxyuridine, the 6-methyl derivatives Ic and Id exhibit neither antibacterial nor antiviral activity. Nor do they exert any antimetabolic effect on the de novo DNA synthesis in primary rabbit kidney cells.

Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene

1988 ◽  
Vol 8 (4) ◽  
pp. 1756-1764
Author(s):  
B Moran ◽  
B Zerler
Keyword(s):  
Dna Synthesis ◽  
Biological Activities ◽  
Rat Kidney ◽  
Kidney Cells ◽  
Transient Expression Assay ◽  
Focus Formation ◽  
Mutant Proteins ◽  
Functional Sites ◽  
Adenovirus E1a ◽  
Cellular Dna

Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products. This deletion did not impede the ability of the E1A products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.

scholarly journals Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH

2006 ◽  
Vol 80 (22) ◽  
pp. 10931-10941 ◽  
Author(s):  
Rachel M. Schowalter ◽  
Stacy E. Smith ◽  
Rebecca Ellis Dutch
Keyword(s):  
Fusion Protein ◽  
Cleavage Site ◽  
Low Ph ◽  
Human Metapneumovirus ◽  
Proteolytic Processing ◽  
Luciferase Reporter ◽  
Transfected Cells ◽  
F Protein ◽  
Viral Fusion Protein ◽  
Glycosylation Sites

ABSTRACT Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

scholarly journals Functional Studies of MLL-AF4 and a Murinized pSer-variant thereof: MLL-AF4 Impairs the Ribosome Biosynthesis Pathway

2021 ◽  
Author(s):  
Anna Lena Siemund ◽  
Eric Kowarz ◽  
Rolf Marschalek
Keyword(s):  
Transcription Factor ◽  
Fusion Protein ◽  
Mammalian Cells ◽  
Biological Activities ◽  
Intracellular Localization ◽  
Biological Behavior ◽  
Hematopoietic Stem ◽  
Functional Studies ◽  
Pol I ◽  
Transcription Factor Complex

Abstract Background: Recent pathomolecular studies on the MLL-AF4 fusion protein revealed that the murinized version of MLL-AF4, the MLL-Af4 fusion protein, was able to induce leukemia when expressed in murine or human hematopoietic stem/progenitor cells (1). In parallel, a group from Japan demonstrated that the pSer domain of the AF4 protein, as well as the pSer domain of the MLL-AF4 fusion is able to bind the Pol I transcription factor complex SL1 (2).Here, we investigated the human MLL-AF4 and a pSer-murinized version thereof for their functional properties in mammalian cells. Gene expression profiling studies were complemented by intracellular localization studies and functional experiments concerning the biological activities in the nuecleolus.Results: Based on our results, we have to conclude that MLL-AF4 is predominantly localizing in the nucleolus, thereby interfering withPol I transcription, and subsequently,also ribosomebiogenesis. The murinized pSer-variant is more localizing in the nucleus, which may explain their different biological behavior. Of note, AF4-MLL is cooperating at the molecular level with MLL-AF4, but not with the pSer-murinized version of it.Conclusion: This study provides new insights and a molecular explanation for the known differences between hMLL-hAF4 (not leukemogenic) and hMLL-mAf4 (leukemogenic). While the human pSer domain is able to efficiently recruit the SL1 transcription factor complex, the murine counterpart is not. This has several consequences for our understanding of t(4;11) leukemia which is by far the most frequent leukemia in infants, childhood and adults suffering from MLL-r acute leukemia.

scholarly journals Development of a one-step immunoassay for triazophos using camel single-domain antibody–alkaline phosphatase fusion protein

2019 ◽  
Vol 411 (6) ◽  
pp. 1287-1295 ◽  
Author(s):  
Kai Wang ◽  
Zhiping Liu ◽  
Guochun Ding ◽  
Ji Li ◽  
Natalia Vasylieva ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
One Step

scholarly journals A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1727-1741 ◽  
Author(s):  
Anuja Krishnan ◽  
Santosh K. Verma ◽  
Prashant Mani ◽  
Rahul Gupta ◽  
Suman Kundu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sendai Virus ◽  
Biological Activities ◽  
Viral Envelope ◽  
Attachment Protein ◽  
Histidine Residues ◽  
Human Parainfluenza Virus ◽  
Mimicking Peptides

ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.

Assessment of the Colorimetric and Fluorometric Assays for Alkaline Phosphatase Activity in Cow's, Goat's, and Sheep's Milk

2008 ◽  
Vol 71 (9) ◽  
pp. 1884-1888 ◽  
Author(s):  
V. KLOTZ ◽  
ART HILL ◽  
K. WARRINER ◽  
M. GRIFFITHS ◽  
J. ODUMERU
Keyword(s):  
Alkaline Phosphatase ◽  
Colorimetric Assay ◽  
Residual Activity ◽  
Raw Milk ◽  
The United States ◽  
Cow’S Milk ◽  
Human Pathogens ◽  
Cow's Milk ◽  
Limit Of Quantitation ◽  
Time Required

Raw milk is a well-established vehicle for the carriage of human pathogens, and many regulatory bodies have consequently mandated compulsory pasteurization as a food safety intervention. The residual activity of alkaline phosphatase (ALP) has historically been used to verify the adequacy of pasteurization of cow's milk. However, there is uncertainty on how the current ALP standards and methods of analysis can be applied to sheep's and goat's milk, which naturally contain different levels of the enzyme than that found in cow's milk. The official ALP methods applied in Canada (colorimetric assay; MFO-3) and in the United States (Fluorophos) were assessed for their ability to detect enzyme activity in raw and pasteurized milk derived from cows, sheep, and goats. The detection limit and the limit of quantitation were 0.8 and 2.02 μg/ml phenol, respectively, for the MFO-3 method and 43 and 85 mU/liter, respectively, for the Fluorophos method. The average ALP levels in raw goat's, cow's, and sheep's milk were 165, 1,562, and 3,512 μg/ml phenol, respectively. Raw milk detection limits, which correspond to raw milk phosphatase levels, were 0.051, 0.485, and 0.023% in cow's, goat's, and sheep's milk, respectively, for the MFO-3 method and 0.007, 0.070, and 0.004%, respectively, for the Fluorophos method. Although both methods can be used for ALP determination in cow's, goat's, and sheep's milk, the Fluorophos assay was superior to the colorimetric MFO-3 method based on sensitivity and time required to complete the analysis.

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