Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.     

Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde.

Liver tissue from miniature pig fetuses was immersion-fixed in fixative mixtures with various concentrations of formaldehyde and glutaraldehyde. The preservation quality of hepatocytes was evaluated ultrastructurally in a peripheral zone (30--130 micron below the surface) and a central zone (500 micron below the surface). In the peripheral zone the best preservation was obtained with a fixative mixture containing 2% formaldehyde and 2% glutaraldehyde and in the central zone with a fixative mixture containing 8% formaldehyde and 8% glutaraldehyde. It is concluded that a better utilization of fairly large tissue blocks for ultrastructural investigation can be obtained by division of the block and subsequent fixation in fixatives containing various concentrations of formaldehyde and glutaraldehyde.     

Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.     

Differentially expressed genes in the liver of thioacetamide treated rats

Thioacetamide is a hepatotoxic and hepatocarcinogenic compound that affects liver metabolism, inhibits mRNA transport and induces enlargement of the nucleolus. To investigate the effect of thioacetamide at the molecular level, differential display RT-PCR was conducted. Analysis of nineteen differentially expressed genes demonstrated that ten cDNAs have their expression inhibited while the other nine were positively affected by thioacetamide. Two of the cDNAs were homologous to known genes-TAP and ankyrin-binding glycoprotein-1, two corresponded to repetitive sequences and seven were homologous to expressed sequence tags. The differential expression of some of the isolated cDNAs was confirmed by northern hybridization. It is proposed that since the product of TAP is involved in mRNA transport, thioacetamide inhibition of TAP expression might, at least partially, explain the thioacetamide-induced swelling of the nucleolus.     

The HIV-1 Rev protein: a model system for coupled RNA transport and translation.

The impact of the Rev protein of the human immunodeficiency virus type 1 (HIV-1) on RNA transport, intranuclear RNA distribution, and gene expression was examined for two Rev-dependent expression systems by means of fluorescence in situ hybridization, immunofluorescence, S1 nuclease protection, and functional assays. In the pgTat expression system, which utilizes authentic HIV-1 splice signals, unspliced mRNA remained entrapped in the nucleus in the absence of Rev and was exported to the cytoplasm in its presence, consistent with published findings. In the pSVAR expression system, significant levels of mRNA were found in the nucleus and cytoplasm in both the presence and absence of Rev, but only in the presence of Rev was mRNA translated into protein. The presence of cytoplasmic untranslated mRNA in the absence of Rev was demonstrated by in situ hybridization analysis of individual cells as well as by S1 nuclease analysis of cell populations. The results indicate that Rev has the potential to affect translation as well as transport, suggesting the possibility that cellular mechanisms exist whereby the translational efficiency of an mRNA may be affected by the manner in which it is transported from the nucleus. Fluorescence hybridization also provided high-resolution visualization of the intranuclear distribution of RNAs containing the Rev response element. This demonstrated for both expression systems that mRNA was not highly localized in tracks or around the nucleolus in the presence or absence of Rev, a nucleolar protein, but was more widely distributed throughout the nucleus. In pgTat transfectants, HIV-1 RNA often became localized in 5 to 20 discrete large intranuclear clusters in the presence of Rev, the potential significance of which is discussed.     

Intrapituitary regulatory system of mammotrophs in the mouse

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor alpha (TGF-alpha) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17beta (E2, 10(-9) M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-alpha antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-alpha mRNA and growth hormone by in situ hybridization and fluorescence-immunocytochemistry demonstrated that TGF-alpha mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-alpha-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-alpha produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.     

Effect of formaldehyde and glutaraldehyde fixation on the immunochemical reactivity of potato virus A with monoclonal antibodies

Potato virus A (PVA) was treated with different glutaraldehyde concentrations at a range of different pH values, and its immunochemical reactivity and integrity was tested in various types of ELISA using a panel of six monoclonal antibodies. Glutaraldehyde fixation was compared with formaldehyde fixation, and there was no significant difference in reactivity between PVA non-treated and treated with formaldehyde, but almost the entire immunochemical activity was lost after treating with glutaraldehyde.     

Effect of Fixation on Demonstration of Phosphatases of Eimeria tenella Grown in Chick Kidney Cell Cultures

SYNOPSIS. The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase > thiamine pyrophosphatase > 5'-nucleotidase > adenosine triphosphatase > acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase. TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase.
Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures.

The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes

A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences.     

Sporicidal Activity of Alkaline Alcoholic Saturated Dialdehyde Solutions

During a search for a sporicidal agent to equal or surpass formaldehyde in activity, a group of saturated dialdehydes ranging from two to six carbons was discovered. These dialdehydes exerted a surprisingly high degree of activity in isopropanol buffered with a carbonate or bicarbonate. Without the proper buffer, practically no activity was observed. A solution of 1% glutaraldehyde, 0.3% NaHCO₃, and 70% isopropanol sterilized stainless-steel penicylinders contaminated with standardized numbers of four spore types in a shorter time than did commercially available 8% formaldehyde solution. Both glyoxal and glutaraldehyde in isopropanol destroyed spores in a relatively short time. Care was taken to eliminate bacteriostasis. Various tests were performed to evaluate accurately experimental and formaldehyde solutions.     

Restrictions on rotational and translational diffusion of pigment in the membranes of a rhabdomeric photoreceptor

Individual, isolated rhabdoms from dark-adapted crayfish (Orconectes, Procambarus) were studied with a laterally incident microbeam that could be placed in single stacks of microvilli. Concentration gradients of metarhodopsin along the lengths of microvilli were produced by local bleaches, accomplished by irradiation with small spots of orange light at pH 9 in the presence of glutaraldehyde or formaldehyde. No subsequent redistribution of pigment was observed in the dark, indicating an absence of translational diffusion. On the basis of comparison with other systems, glutaraldehyde, but not formaldehyde (0.75%), would be expected to prevent diffusion of protein in the membrane. Under the same conditions photodichroism is observed, indicating an absence of free Brownian rotation. Photodichroism is larger in glutaraldehyde than in formaldehyde, suggesting that the bifunctional reagent quiets some molecular motion that is present after treatment with formaldehyde. Quantitative comparison of photodichroism with mathematical models indicates that the pigment absorption vectors are aligned within +/- 50 degrees of the microvillar axes and are tilted into the surface of the membrane at an average value of about 20 degrees. The photoconversion of rhodopsin to metarhodopsin is accompanied by an increase in molar extinction of about 20% at the lambda maxand a reorientation of the absorption vector by several degrees. The transition moment either tilts further into the membrane or loses some of its axial orientation, or both. The change in orientation is 3.5 time larger in formaldehyde than in glutaraldehyde.     

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia

A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15-20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.     

The nucleolar localisation signal of the HTLV-I protein p27rex is important for stabilisation of IL-2 receptor alpha subunit mRNA by p27rex

In this study we investigated the mechanism of stabilisation of IL-2 receptor alpha subunit mRNA by the HTLV-I protein p27rex. We tested the role of the nucleolar targetting signal in rex by introducing mutations. Three deletion mutants could not express rex protein in the nucleolus and although protein was still expressed in the nucleoplasm none of the mutants could stabilise IL-2R alpha mRNA. A substitution mutant could be expressed in the nucleolus and could also stabilise IL-2R alpha mRNA. The data show that the nucleolar targetting signal is crucial for stabilisation of IL-2R alpha mRNA by rex and raise the possibility that transport of mRNA from nucleus to cytoplasm can involve the nucleolus.     

A study of staining for electron microscopy using collagen as a model system—VII. The effect of formaldehyde fixation

Exposure to formaldehyde brings about small but readily detectable changes in the staining behaviour of collagen fibrils. These changes can be interpreted in chemical terms by comparing fibril staining patterns with artificial patterns computer-generated from sequence data. Positive staining with phosphotung-state (where heavy metal is confined to anions), shows that most of the lysyl and hydroxylysyl side-chains lose their charge character as a result of formaldehyde treatment and cease to take up staining ions. The charge character of arginyl (and probably histidyl) residues is unaltered and these residues continue to react with stain. Acidic residues are also unaffected. These results accord with biochemical evidence that the initial reaction between proteins and formaldehyde leading to subsequent cross-linking involves modification of ε-amino (and α-amino) groups. They show too that the secondary condensation producing the actual cross-link does not alter the charge character of the second group, at least when it is on an arginyl (or histidyl) side-chain.Formaldehyde-induced changes in stain deposition can also be detected after negative staining, although they are slight compared with those brought about by glutaraldehyde. Unlike glutaraldehyde, formaldehyde introduces no bulky polymeric adducts into the fibril structure, and the conspicuous stain-excluding bands seen in negative staining patterns following glutaraldehyde fixation are absent after exposure to formaldehyde. For this reason, where chemical fixation is used to stabilize macromolecules and supramolecular aggregates prior to negative staining and high resolution electron optical imaging, formaldehyde would seem to be preferable to glutaraldehyde. Data from fibril staining patterns and from thermal stability measurements (made on collagen gels) show that formaldehyde fixation does not preclude a subsequent reaction with glutaraldehyde.As with other fixatives, there is reduced accessibility to stain after formaldehyde treatment. Accessibility is least in the overlap zone where the denser packing of collagen molecules provides greater opportunities for intermolecular cross-linking. Gel electrophoresis confirms that formaldehyde-induced cross-links in fibrils are predominantly intermolecular.