Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues

Summary Proliferating cell nuclear antigen is expressed in cells from late G₁ through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.     

Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.Understanding fundamental biological processes relies on probing intra- and extracellular environments, targeted delivery inside living cells and tissues, and real-time detection and imaging of chemical markers and biomolecules (1,2). Typically, information about molecules in cellular environments is obtained by fluorescence microscopy (3). This is a powerful imaging tool for localizing and imaging samples but requires fluorescent labels and markers and lacks capabilities for quantitative mapping of the chemical composition in complex systems. In this regard, confocal Raman spectroscopic imaging is becoming increasingly popular for label-free chemical detection, due to the inherent scattering nature of all biomolecules (4,5). However, confocal Raman imaging alone does not allow live, high-resolution imaging of larger regions of interest in complex biological tissues. Transcutaneous Raman spectroscopy has the potential as a tool for in vivo bone quality assessment (6), whereas the time- and space-resolved Raman spectroscopy allows the visualization in vivo of the distributions of molecular species in human and yeast cells (4,5,7). Here we developed a correlative Raman and fluorescence imaging method that combines the strengths and compensates for the shortcomings of each of these imaging modalities and allows studying in vivo processes in complex animal models such as zebrafish larvae. There are two main advantages of this approach over previous studies (8,9): low light intensity and high acquisition rate, making it well suited for real-time investigation of live samples.Fig. 1, a and b, shows a schematic representation of the experimental setup and of the optical path, respectively. The two techniques are implemented on a commercially available Raman microscope body to perform simultaneously confocal Raman spectroscopy and wide-field fluorescence imaging (see the Supporting Material for details of components). Briefly, the multimodality of the setup is provided by a combination of dichroic mirrors (DM 1–3) and filters that at turns reflect or transmit the excitation and emission signals. This combination of optics allows simultaneous collection of fluorescence images (2560 × 2160 pixels at 30 fps) with excitation at 400 and 490 nm and spatially resolved Raman spectra with excitation at 633 nm.Open in a separate windowFigure 1Fluorescence imaging of zebrafish larvae. (a) Cartoon of the experimental setup showing how the different modules are assembled onto the microscope for the simultaneous use of confocal Raman spectroscopy and fluorescence imaging. (b) Schematic representation of the optical path. (c) Fluorescence image of calcium-containing tissues, and fluids stained with calcein blue and excited at 400 nm (top). Endothelial cells of transgenic tg(fli1:EGFP)y1 zebrafish excited at 490 nm (bottom).As a proof of principle, we have studied the different mineral phases involved in bone formation of the zebrafish larvae. The bone development process involves the transport of ions to specific cells (osteoblasts) that are responsible for the subsequent mineral formation and deposition. The mineral phase in these cells is a poorly characterized disordered calcium phosphate (10–12). The mineral-bearing intracellular vesicles release their content into the extracellular collagen fibrils, where the mineral subsequently crystallizes as carbonated hydroxyapatite (13). Very little is known about the phase transformations the mineral undergoes after the deposition into the collagen matrix in vivo. Raman spectroscopy studies of bone tissue in organ cultures evidenced that the inorganic mineral deposition proceeds through transient intermediates including octacalcium phosphate-like (OCP) minerals (14).To assess the feasibility of imaging a vertebrate organism, fluorescence images of an entire zebrafish larva (Fig. 1 c) were acquired with the correlative fluorescence-Raman setup. The two images in Fig. 1 c were composed by merging several low-magnification (10×) fluorescence images. Larvae of transgenic zebrafish Tg(fli:EGFP); nac mutants (albino fish) expressing EGFP in the cytoplasm of endothelial cells was used. The newly formed bones were stained by soaking the live embryo noninvasively in the calcium markers calcein blue 0.2% wt or calcein green 0.2% wt.The calcein blue marker is excited at 400 nm. It is labeling bones and can be also detected as a fluorescent marker not associated with formed bones (e.g., stomach) (Fig. 1 c, top). At 490 nm, calcein green and endothelial cells within blood vessels expressing EGFP are excited (Fig. 1 c, bottom). Because EGFP and calcein blue have significantly different excitation and emissions spectra, dual staining with calcein blue (as a mineral marker) and EGFP allows fast-switching dual-wavelength fluorescence imaging. Furthermore, because the spectra of the calcium markers and EGFP do not extend beyond the Raman laser, these fluorophores are appropriate candidates for experiments requiring Raman and fluorescence imaging. The dual-excitation offers the capability of mapping several tissues in a single experiment at the video rate. This, in principle, could be used to probe different parameters of the microenvironment (e.g., pH (15), temperature (16), viscosity (17), and calcium concentration (18)) using wavelength-ratiometric fluorescence imaging which, in correlation with confocal Raman spectroscopy, could open new strategies in studies of the microenvironmental properties in living tissues.The fin rays of zebrafish are a simple, growing bone-model system, in which the fins are gradually mineralized within spatially resolved regions (19). Raman spectroscopy revealed details of the calcein green-stained fin where new bone is deposited (Fig. 2). In Fig. 2 a, a fluorescence image of a zebrafish larva analogous to the top image in Fig. 1 c is shown. The right inset in Fig. 3 b shows higher-magnification (60× water-immersion objective) details of the calcein green-stained fin typical of newly deposited bone. Raman spectra of progressively mineralized bone tissue were acquired within representative regions (Fig. 2 b; numbered 1–4). The spectra exhibit characteristic bands that can be assigned to the organic protein extracellular matrix (amide I, amide III, Phe, C-H, etc.) and the inorganic mineral content (v₁, v₂, v₄ of PO₄³⁻).Open in a separate windowFigure 2Correlative fluorescence-Raman imaging of zebrafish fin bone maturation. (a) Low-resolution (10×) fluorescence image of zebrafish stained with calcein green, with high-resolution (60×) details (right inset in panel b) of a representative fin ray region where Raman spectra (b) of progressively mineralized bone tissue were acquired (numbered 1–4). (Left inset in panel b) Integral of the orientation independent mineral band (v₂) where a clear drop of the mineral content can be observed.The analyses of the orientation-independent v₂ phosphate band revealed a clear drop in the mineral content based on the intensity integral (left inset in Fig. 2 b). Assuming that the spectrum collected in region 4 contains only organic matrix (very small phosphate-related peaks) and by subtracting it from the spectrum of mineral-rich bone region (spectrum 1, proximal part of the tail bone), spectral features of only the mineral phase can be plotted (black line). In addition to the phosphate (PO₄³⁻) and carbonate (CO₃²⁻) bands assignable to the carbonated apatite phase characteristic of the more mature bone mineral, several peaks related to the hydrogen phosphate (HPO₄²⁻) species can be clearly distinguished.The HPO₄²⁻ peaks are characteristic of the OCP mineral phase that has been postulated, together with amorphous calcium phosphate, as an intermediate mineral phase in the process of bone maturation (10,13,14,20), but never observed directly in living animals. Our findings show in vivo potential of the correlative setup envisioned by Crane et al. (14) and confirm that the mineral maturation indeed proceeds through an OCP-like mineral phase. Further analysis of the mineral spectrum in Fig. 2 b reveals an extremely broad band in the region 800–1100 cm⁻¹. This envelope can be related to hydrogenated phosphate species typical of amorphous calcium phosphate precipitated in an acidic environment (see Fig. S1 in the Supporting Material), suggesting that this phase is also contributing to the maturation process.In conclusion, the methodology developed here allows for unprecedented chemical characterization of fluorescently-labeled biological tissues in vivo. The approach is suitable for long-term in vivo characterization of zebrafish bone mineralization under (patho-)physiological conditions. Furthermore, the setup can be upgraded to host other advance fluorescence imaging techniques such as super-resolution microscopy (e.g., photoactivated localization microscopy), two-photon excitation, and Forster resonance energy transfer or fluorescence lifetime imaging microscopy, and be applied on both in vivo and in vitro specimens. This opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces.     

Calcium concentration threshold and translocation kinetics of EGFP-DOC2B expressed in cultured Aplysia neurons

The double C2 domain protein family (DOC2) is characterized by two calcium-binding domains (C2). Upon binding to calcium, the affinity of the protein to phospholipids is significantly increased, leading to translocation of the protein from the cytosol to the plasma membrane. These properties, and the binding domain of DOC2B to Munc13, suggested that DOC2B could play a role in augmentation and potentiation of synaptic release. Nevertheless, the level of the free intracellular calcium concentration ([Ca(2+)](i)) which triggers its translocation under in vivo conditions, is not known. Using cultured Aplysia neurons that express rat EGFP-DOC2B, we found that the [Ca(2+)](i) increment necessary to induce EGFP-DOC2B translocation is approximately 200 nM in the bulk of the cytoplasm. The rate of EGFP-DOC2B recruitment to the plasma membrane is slower than the [Ca(2+)](i) elevation rate, while the detachment of EGFP-DOC2B from it is faster than the calcium removal. The extent of EGFP-DOC2B translocation to the plasma membrane reflects local submembrane [Ca(2+)](i). Our observations are consistent with the view that DOC2B can participate in the regulation of neurotransmitter release. It should be noted that EGFP-DOC2B could be used as a tool to map sub-membrane calcium dynamics under physiological conditions.     

Altered phosphorylation of lung proteins following administration of butylated hydroxytoluene (BHT) to mice.

The incorporation of P³² from (γ?³²P)ATP into lung proteins was examined using lung extracts from mice which had been injected with BHT. Increased phosphorylation, relative to mice not treated with BHT, was associated with proteins of 87,000 M.W. (5–12 fold) and 135,000 M.W. (40–70%). These changes in phosphorylation correlated in time with the transient lung enlargement induced by BHT and were dependent upon the dose of BHT. Antioxidants and structural derivatives of BHT which did not affect lung size also had no effect on lung protein phosphorylation. BHT slightly increased cyclic AMP-dependent protein kinase activity but had no effect on cyclic AMP-binding activity.     

Zebrafish as a Model for Monocarboxyl Transporter 8-Deficiency

Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.     

Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.     

Strain-specific postnatal changes in the activity and tissue levels of protein kinase C

The specific activity of protein kinase C from adult mouse lung and spleen was higher than in the corresponding tissues from neonatal mice. BALB/cBy mice had higher lung and spleen protein kinase C activities at both ages than did A/J mice, and the extent of this strain difference increased with age. These activity differences reflected the tissue levels of the 80 kD form of protein kinase C, as determined by quantitative immunoblotting. These genetic and ontogenetic differences provide an interesting model with which to study the regulation of protein kinase C gene expression.     

Pattern analysis in successional communities – An approach for studying shifts in ecological interactions

Abstract. Many ecological studies have addressed issues of vegetation spatial patterns in attempts to understand the processes generating them. We investigated changes in ecological processes during succession via the analysis of shrubs’ spatial patterns in a system of linear sand dunes, an arid ecosystem located in the Negev Desert in Israel during three consecutive years. We hypothesized that spatial patterns change from clustered to regular as succession progresses due to changes in the relative importance of facilitation and competition in this environment. In this ecosystem communities of early successional stages are frequently disturbed by high rates of sand movement, whereas in later successional stages sand stability is high. We mapped in the field individual shrubs on high‐resolution aerial photographs, and converted the digital images to a GIS data set. Using Ripley's K‐function we analysed spatial patterns at three levels: the single‐species level, among species and at the individual level, in three communities characterizing different successional stages. In the early successional communities we found clustered spatial patterns, in comparison with stable habitats where spatial patterns tended to be regular. We argue that these shifts in spatial patterns are indicative of the assumption that in this sand‐dune system ecological interactions change from facilitation to competition as succession progresses. Further, we argue that these interactions operate in different spatial scales at the different successional stages, and that the study of these processes should be conducted at the spatial scales specific to each community.