Localization of luteinizing hormone β-mRNA by in situ hybridization in the sheep pars tuberalis

Summary The localization of luteinizing hormone beta (LH)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded ³²P-or ³⁵S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by using a specific LH-antiserum indicated that the labelling of LH-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH subunit, but that the level of translation greatly varies according to the location of the cells.     

Cellular localization of induced human interferon-β mRNA by non-radioactive in situ hybridization

Summary Induced interferon- (IFN-) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN- mRNA was transiently induced by poly r(I):r(C) in fibroblasts 2–4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN- mRNA with a maximum at 4–8 h. The kinetics of the IFN- mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern biotting experiments of total cellular RNA.     

Meiotic pairing in wheat-rye derivatives detected by genomic in situ hybridization and C-banding — A comparative analysis

Meiosis of triticalextetraploid rye hybrids (genome constitution ABRRR) was analysed by genomic in situ hybridization (GISH) and C-banding. The results obtained reveal a considerable difference between these techniques with regard to their efficiency in detecting any type of pairing, either homologous or homoeologous. Thus the percentage of pollen mother cells containing wheat/rye homoeologous associations determined by C-banding and GISH was 2.5 and 9.2, respectively. Such a discrepancy can be ascribed to a certain proportion of wheat/rye associations not being identified by C-banding. The potential and limitations of the two techniques for meiotic analysis are discussed.     

Improved paclitaxel production by in situ extraction and elicitation in cell suspension cultures of Taxus chinensis

Dibutyl phthalate, oleic acid and terpineol were used to extract paclitaxel in situ fromTaxus chinensis suspension cultures. Oleic acid/terpineol (1:1, v/v) added to the cultures gave a higher paclitaxel concentration, compared with either of them alone. Oleic acid/terpineol (1:1, v/v) incorporated into the cultures at 3:50 (v/v) 4 days after elicitation, which was carried out by adding 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ to 10-day-old cultures, resulted in the greatest paclitaxel production of 48 mg l^–1 at day 10 after elicitation. This was double that of the culture by elicitation, and 7-fold higher than that of the culture by in situ extraction.     

Regional mapping of the human gene for lysosomal α-glucosidase by in situ hybridization

Summary The current approach to the chromosomal localization of genes coding for lysosomal enzymes has been the correlation of enzymatic and karyotypic analyses of human-rodent somatic cell hybrids. The feasibility of regional mapping depends on the availability of human cells with informative chromosomal rearrangements. In this communication we report the first localization of a gene coding for a lysosomal enzyme by in situ hybridization. The application of an acid -glucosidase cDNA probe to normal human chromosomes allowed direct regional mapping of the -glucosidase locus (GAA) to the region q23q25 of chromosome 17.     

Comparison of various permeabilization treatments on Kluyveromyces by determining in situ β-galactosidase activity

Permeabilization treatments using organic solvents or physical methods were applied to Kluyveromyces bulgaricus and compared by measuring the β-galactosidase activity of whole cells. The minimum solvent concentrations to be used for obtaining a good permeabilization were: 10% n-butanol; 20% propanol; 30% isopropanol, tert-butanol; 40% ethanol, acetone and 70% dimethylsulphoxide. Toluene/ethanol (1 : 4) at 10% was less effective. The addition of the surfactant Brij 35 to lower alkanol concentrations did not bring about a significant permeabilization but the treatment of cells with Brij 35 with a small amount of toluene in ethanol (4 : 96) resulted in a high enzymatic activity. Yeast pellet, but not yeast suspension, submitted to five cycles of freezing and thawing displayed an enzymatic activity similar to those obtained by organic solvents.     

Assignment of human β-galactosidase-A gene to 3p21.33 by fluorescence in situ hybridization

GM₁ gangliosidosis and Morquio syndrome type B (MPS IVB) are inherited lyosomal storage disorders associated with deficiency of -galactosidase-A (GAL_A) activity. A recombinant plasmid containing a biotinylated cDNA (2.4-kb insert) encoding human GAL_A was used to localize the enzyme locus by fluorescence in situ hybridization (FISH). The human GAL_A gene was assigned to 3p21.33 by FISH.     

Localization of type 5 17β-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization

The mouse enzyme type 5 17-hydroxysteroid dehydrogenase (17-HSD) catalyzes the conversion of androstenedione to testosterone and, to a lesser degree, the conversion of estrone to estradiol. In order to determine the exact sites of action of type 5 17-HSD, we studied the cellular localization of the mRNA of the enzyme in mouse tissues by using in situ hybridization. Specific hybridization signal was found in the liver, ovary, adrenal cortex, and kidney. In the liver of mice of both sexes, a strong signal was observed in all hepatocytes. In the ovary, specific labeling was detected in the granulosa and theca interna cells in growing follicles and in luteal cells. In the female adrenal cortex, intense labeling was restricted to the zona reticularis, whereas no type 5 17-HSD mRNA expression could be found in the male adrenal cortex. In the kidney of mice of both sexes, type 5 17-HSD mRNA was expressed in epithelial cells in both the proximal and distal convoluted tubules. The data indicate that androgens and estrogens are formed via the action of type 5 17-HSD in specific cell types in the liver, ovary, adrenal cortex, and kidney.This work was supported by Genome Canada and Genome Québec.     

Three major mesoplanktonic communities resolved by in situ imaging in the upper 500 m of the global ocean

Aim

The distribution of mesoplankton communities has been poorly studied at global scale, especially from in situ instruments. This study aims to (1) describe the global distribution of mesoplankton communities in relation to their environment and (2) assess the ability of various environmental-based ocean regionalizations to explain the distribution of these communities.

Location

Global ocean, 0–500 m depth.

Time Period

2008–2019.

Major Taxa Studied

Twenty-eight groups of large mesoplanktonic and macroplanktonic organisms, covering Metazoa, Rhizaria and Cyanobacteria.

Methods

From a global data set of 2500 vertical profiles making use of the Underwater Vision Profiler 5 (UVP5), an in situ imaging instrument, we studied the global distribution of large (>600 μm) mesoplanktonic organisms. Among the 6.8 million imaged objects, 330,000 were large zooplanktonic organisms and phytoplankton colonies, the rest consisting of marine snow particles. Multivariate ordination (PCA) and clustering were used to describe patterns in community composition, while comparison with existing regionalizations was performed with regression methods (RDA).

Results

Within the observed size range, epipelagic plankton communities were Trichodesmium-enriched in the intertropical Atlantic, Copepoda-enriched at high latitudes and in upwelling areas, and Rhizaria-enriched in oligotrophic areas. In the mesopelagic layer, Copepoda-enriched communities were also found at high latitudes and in the Atlantic Ocean, while Rhizaria-enriched communities prevailed in the Peruvian upwelling system and a few mixed communities were found elsewhere. The comparison between the distribution of these communities and a set of existing regionalizations of the ocean suggested that the structure of plankton communities described above is mostly driven by basin-level environmental conditions.

Main Conclusions

In both layers, three types of plankton communities emerged and seemed to be mostly driven by regional environmental conditions. This work sheds light on the role not only of metazoans, but also of unexpected large protists and cyanobacteria in structuring large mesoplankton communities.     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.     

Human δ-aminolevulinate dehydratase: chromosomal localization to 9q34 by in situ hybridization

Summary The structural gene for human -aminolevulinate dehydratase (ALA-D) has been localized to chromosomal region 9q34 by in situ hybridization using a [¹²⁵I]-labeled human -aminolevulinate dehydratase cDNA. Of the 150 silver grains analyzed, 25% were localized to chromosome 9q, while 12% and 8% were on chromosomes 1p and 13q, respectively. The single chromosomal region q34 had over 90% of the total grains observed on chromosome 9. In contrast, the grains on chromosomes 1p and 13q were dispersed, consistent with the absence of any human ALD-D pseudogenes. Southern blot analysis of somatic cell hybrids informative for ALA-D (Wang et al. 1985) also was consistent and supported the finding of only one locus for this heme biosynthetic enzyme.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

Specific in situ discrimination of amyloid fibrils versus α-helical fibres by the fluorophore NIAD-4

A wide range of human pathologies, including neurodegenerative diseases and other forms of amyloidosis, are associated with the formation of insoluble fibrillar protein aggregates known as amyloids. To gain insights into this process analytical methods are needed, which give quantitative data on the molecular events that are taking place. The dye Thioflavin T (ThT) is widely used for the spectroscopic determination of amyloid fibril formation. Different binding affinities to amyloids at neutral and acidic pH and the frequently observed poor binding at acidic pH are problematic in the use of the cationic ThT. The uncharged fluorescence probe [[5'-(4-hydroxyphenyl)[2,2'-bithiophen]-5-yl]methylene]-propanedinitrile (NIAD-4) has been recently designed by Swager and coworkers, in order to eliminate some of the limitations of ThT. Here we have used this novel dye for in vitro monitoring of the amyloid formation processes of de novo designed model peptides. Amyloid structures were successfully detected by NIAD-4 at neutral as well as acidic pH and no significant fluorescence was detectable in the presence of α-helical fibres. Thus, NIAD-4 proved to be a valuable alternative to ThT for spectroscopic studies on amyloid structures over a broad pH range.     

Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol–alkyne addition

Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the observed covalent adduct formation. We therefore propose that the reaction is an in situ thiol–alkyne addition. Due to the high chemical inertness of the alkyne moiety the respective protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was observed in lysates of cell lines expressing the cognate proteases after transient transfection.     

Root growth in biopores—evaluation with in situ endoscopy

Background and aims

The significance of biopores for nutrient acquisition from the subsoil depends on root-soil contact, which in turn is influenced by root architecture. The aim of this study was to detect differences regarding the architecture and root-soil contact of homorhizous barley roots (Hordeum vulgare L.) and allorhizous oilseed rape roots (Brassica napus L.) growing in biopores.

Methods

In situ endoscopy was used as a technique that allows non-destructive display of pore wall characteristics and root morphology inside large biopores under field conditions.

Results

For both crops, about 85 % of all roots did establish contact to the pore wall. However, according to their different root architecture, the two crops varied in their strategy of resource acquisition: While barley was characterized by thin vertical or ingrowing roots, most of them in direct contact to the pore wall, oilseed rape established contact to the pore wall predominantly via lateral roots.

Conclusions

Root morphological and pore wall assessment with in situ endoscopy in combination with detailed studies of soil biochemical and soil physical parameters of the pore wall is considered an essential prerequisite for more precise future modelling of nutrient acquisition and uptake.     

Biodiversity and bamboo genetic resources in Asia:in situ, community-based and ex situ approaches to conservation

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Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.     

Distribution of β2-like adrenergic receptors in the cnidarian Renilla koellikeri as revealed by autoradiography and in situ hybridization

Autoradiography and in situ hybridization were used to examine the histological distribution of the previously characterized 2-like adrenergic receptors involved in the bioluminescent activity of the sea pansy Renilla koellikeri. The use of [³H]-(±)CGP12177 as radiologand revealed autoradiographic labelling of the refringent granule-filled endoderm at the base of autozooid tentacles and autozooid columns, and in the corresponding endoderm of siphonozooid polyps, all areas where photocytes are concentrated. The presence of excess (10 M) unlabelled (±)CGP12177 or atenolol in the incubation mixture substantially reduced total [³H]-(±)CGP12177 labelling. Under low stringency hybridization washing, human 2-adrenoceptor oligonucleotide probe signals were detected in granular cells located in those areas of polyp endoderm that were labelled by [³H]-(±)CGP12177. These cells were previously shown to be distinct from, but in close proximity to photocytes. No other cell or tissue type was labelled in polyps or throughout colonial tissues. The results suggest that a conserved form of 2-adrenergic receptors is present and synthesized in a unique type of endodermal cell indirectly involved in sea pansy bioluminescence control.     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.