In situ localization of the specific cell-binding fragment on the sponge aggregation factor

1986 ◽  
Vol 95 (1-3) ◽  
pp. 108-112 ◽  
Author(s):  
Monika Gramzow ◽  
Bärbel Diehl-Seifert ◽  
Thomas Zaubitzer ◽  
Werner E.G. Müller
Keyword(s):  
Specific Cell ◽  
Cell Binding ◽  
Aggregation Factor

scholarly journals Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.

1986 ◽  
Vol 102 (4) ◽  
pp. 1344-1349 ◽  
Author(s):  
M Gramzow ◽  
M Bachmann ◽  
G Uhlenbruck ◽  
A Dorn ◽  
W E Müller
Keyword(s):  
Single Cells ◽  
Sodium Dodecyl ◽  
Cell System ◽  
Specific Cell ◽  
Cell Binding ◽  
Aggregation Factor ◽  
Indirect Immunofluorescence Staining ◽  
Number Of Binding Sites ◽  
Ca Ions

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.

Temporal changes in the expression of the insulin-like growth factor II gene associated with tissue maturation in the human fetus

Development ◽  
1989 ◽  
Vol 106 (3) ◽  
pp. 543-554 ◽  
Author(s):  
A.L. Brice ◽  
J.E. Cheetham ◽  
V.N. Bolton ◽  
N.C. Hill ◽  
P.N. Schofield
Keyword(s):  
Growth Factor ◽  
Cell Types ◽  
Specific Cell ◽  
Insulin Like Growth Factor ◽  
Vitro Fertilization ◽  
Age Related ◽  
Factor Ii ◽  
Metanephric Blastema

The insulin-like growth factors are broadly distributed in the human conceptus and are thought to play a role in the growth and differentiation of tissues during development. Using in situ hybridization we have shown that a wide variety of specific cell types within tissues express the gene for insulin-like growth factor II at times of development from 18 days to 14 weeks of gestation. Examination of blastocysts produced by in vitro fertilization showed no expression, thus bracketing the time of first accumulation of IGF-II mRNA to between 5 and 18 days postfertilization. The pattern of IGF-II expression shows specific age-related differences in different tissues. In the kidney, for example, expression is found in the cells of the metanephric blastema which is dramatically reduced as the blastema differentiates. The reverse is also seen, and we have noted an increase in expression of IGF-II in the cytotrophoblast layer of the placenta with gestational age. The sites of expression do not correlate with areas of either high mitotic activity or specific types of differentiation, but the observed pattern of expression in the kidney, adrenal glands and liver suggests an explanation for the abnormally high IGF-II mRNA expression in developmental tumours such as Wilms' tumour.

scholarly journals Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

1993 ◽  
Vol 41 (7) ◽  
pp. 1023-1030 ◽  
Author(s):  
R Gold ◽  
M Schmied ◽  
G Rothe ◽  
H Zischler ◽  
H Breitschopf ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Specific Cell ◽  
Nick Translation ◽  
Proliferating Cells ◽  
Cell Surface Antigens ◽  
Simultaneous Application ◽  
Tissue Sections ◽  
In Situ Nick Translation

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

scholarly journals Methods to Enhance Signal Using Isotopic In Situ Hybridization

2002 ◽  
Vol 50 (8) ◽  
pp. 1031-1037 ◽  
Author(s):  
Betty Ky ◽  
Paul J. Shughrue
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Oxytocin Receptor ◽  
Specific Cell ◽  
Rat Hypothalamus ◽  
Low Levels ◽  
Tuberoinfundibular Peptide ◽  
Cryostat Sections

Isotopic in situ hybridization (ISH) has been established as a uniquely powerful tool for the study of gene expression in specific cell types. This technique allows the visualization and quantification of gene expression and gene expression changes in cells. In our study of biological and molecular phenomena, we have increasingly encountered the need to detect small changes in gene expression as well as genes of low abundance, such as the oxytocin receptor (OTR) and the tuberoinfundibular peptide of 39 residues (Tip39). To increase the sensitivity of isotopic ISH for detection of rare mRNAs, we performed ISH on cryostat sections of rat hypothalamus and thalamus with 35S-labeled riboprobes and amplified the signal by hybridizing over 2 nights as well as labeling the probe with both [35S]-UTP and [35S]-ATP. These two methods of enhancement independently and in combination demonstrated a dramatic increase in signal, allowing the visualization of low levels of gene expression previously undetectable by conventional methods.

scholarly journals Marine Planktonic Archaea Take Up Amino Acids

2000 ◽  
Vol 66 (11) ◽  
pp. 4829-4833 ◽  
Author(s):  
Cleber C. Ouverney ◽  
Jed A. Fuhrman
Keyword(s):  
Amino Acids ◽  
In Situ Hybridization ◽  
Cell Types ◽  
Similar Proportion ◽  
Specific Cell ◽  
Natural Conditions ◽  
The Pacific ◽  
The Pacific Ocean ◽  
Archaeal Communities

ABSTRACT Archaea are traditionally thought of as “extremophiles,” but recent studies have shown that marine planktonic Archaea make up a surprisingly large percentage of ocean midwater microbial communities, up to 60% of the total prokaryotes. However, the basic physiology and contribution of Archaea to community microbial activity remain unknown. We have studied Archaea from 200-m depths of the northwest Mediterranean Sea and the Pacific Ocean near California, measuring the archaeal activity under simulated natural conditions (8 to 17°C, dark and anaerobic) by means of a method called substrate tracking autoradiography fluorescence in situ hybridization (STARFISH) that simultaneously detects specific cell types by 16S rRNA probe binding and activity by microautoradiography. In the 200-m-deep Mediterranean and Pacific samples, cells binding the archaeal probes made up about 43 and 14% of the total countable cells, respectively. Our results showed that the Archaea are active in the uptake of dissolved amino acids from natural concentrations (nanomolar) with about 60% of the individuals in the archaeal communities showing measurable uptake. Bacteria showed a similar proportion of active cells. We concluded that a portion of these Archaea is heterotrophic and also appears to coexist successfully with Bacteria in the same water.

Expression and cellular localization of substance P/neurokinin A and neurokinin B mRNAs in the rat retina

1989 ◽  
Vol 3 (6) ◽  
pp. 527-535 ◽  
Author(s):  
Nicholas C. Brecha ◽  
Catia Sternini ◽  
Karl Anderson ◽  
James E. Krause
Keyword(s):  
Substance P ◽  
Ganglion Cell ◽  
Cellular Localization ◽  
Amacrine Cells ◽  
Specific Cell ◽  
Neurokinin A ◽  
Neurokinin B ◽  
Rat Retina ◽  
Rna Probes

AbstractThe mammalian tachykinin peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) are encoded by distinct mRNAs derived from separate preprotachykinin (PPT) genes. The SP/NKA-encoding PPT gene generates three mRNAs by alternative RNA processing: α-PPT mRNA, which encodes SP only, and β- and γ-PPT mRNAs, which encode both SP and NKA. The NKB-encoding PPT gene generates mRNAs that produce NKB. The distribution and cellular localization of SP, NKA and NKB mRNAs in the rat retina were studied by RNA blot and in situ hybridization techniques. Blot hybridization analysis of retinal RNA extracts with [32P]-labeled RNA probes complementary to SP/NKA and NKB mRNAs demonstrated single bands of hybridization at 1300 and 900 bases, respectively. Solution hybridization-nuclease protection experiments showed multiple SP/NKA-encoding transcripts with relative levels of ρ-PPT mRNA > β-PPT mRNA ≫ α-PPT mRNA. In situ hybridization histochemistry with [35S]-labeled antisense RNAs demonstrated thatSP/NKA-encoding transcripts are expressed in small-to-medium somata located in the proximal inner nuclear, inner plexiform, and ganglion cell layers, whereas NKB-encoding transcripts are expressed in small-to-medium somata located only in the ganglion cell layer. In this layer, cells containing NKB mRNAs are more numerous than those containing SP/NKA mRNAs. Only background labeling was observed in sections incubated with sense RNA probes, pretreated with RNase A prior to hybridization or incubated in hybridization buffer without the labeled probe. Immunohistochemical studies with a monoclonal antibody directed to the conserved COOH-terminal sequence of the tachykinin peptides revealed tachykinin-like immunoreactive somata with similar size and distribution to those containing SP/NKA- and NKB-encoding transcripts. These results indicate that both SP/NKA and NKB mRNAs are present in the rat retina and that the PPT genes are differentially expressed in specific cell populations. The size and distribution of these cells suggest that they are amacrine and displaced amacrine cells, however, the possibility that tachykinins are present also in ganglion cells in the rat retina cannot be ruled out.

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