Identification of 5-Bromo-2’-Deoxyuridine-Labeled Cells during Mouse Spermatogenesis by Heat-Induced Antigen Retrieval in Lectin Staining and Immunohistochemistry

DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2’-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.     

Differential Modulation of ��- and ��-Opioid Receptor Agonists by Endogenous RGS4 Protein in SH-SY5Y Cells

Regulator of G-protein signaling (RGS) proteins are a family of molecules that control the duration of G protein signaling. A variety of RGS proteins have been reported to modulate opioid receptor signaling. Here we show that RGS4 is abundantly expressed in human neuroblastoma SH-SY5Y cells that endogenously express μ- and δ-opioid receptors and test the hypothesis that the activity of opioids in these cells is modulated by RGS4. Endogenous RGS4 protein was reduced by ∼90% in SH-SY5Y cells stably expressing short hairpin RNA specifically targeted to RGS4. In these cells, the potency and maximal effect of δ-opioid receptor agonist (SNC80)-mediated inhibition of forskolin-stimulated cAMP accumulation was increased compared with control cells. This effect was reversed by transient transfection of a stable RGS4 mutant (HA-RGS4C2S). Furthermore, MAPK activation by SNC80 was increased in cells with knockdown of RGS4. In contrast, there was no change in the μ-opioid (morphine) response at adenylyl cyclase or MAPK. FLAG-tagged opioid receptors and HA-RGS4C2S were transiently expressed in HEK293T cells, and co-immunoprecipitation experiments showed that the δ-opioid receptor but not the μ-opioid receptor could be precipitated together with the stable RGS4. Using chimeras of the δ- and μ-opioid receptors, the C-tail and third intracellular domain of the δ-opioid receptor were suggested to be the sites of interaction with RGS4. The findings demonstrate a role for endogenous RGS4 protein in modulating δ-opioid receptor signaling in SH-SY5Y cells and provide evidence for a receptor-specific effect of RGS4.μ- and δ-opioid receptors are members of the G protein-coupled receptor family and interact with Gα_i/o proteins (1, 2). This results in signaling to a variety of downstream effectors, including adenylyl cyclase and the mitogen-activated protein kinase (MAPK)² cascade. Signaling of opioid receptors is regulated negatively by regulator of G protein signaling (RGS) proteins (3, 4). These are a family of molecules containing a “RGS consensus” domain that bind to Gα subunits and act as GTPase-accelerating proteins to increase the rate of GTP hydrolysis. This results in a decrease in the lifetime of the active Gα-GTP and free Gβγ subunits and limits signaling to downstream effectors (5–8). The mechanisms by which RGS proteins selectively modulate G protein-mediated receptor signal transduction pathways, especially opioid receptor signaling, are beginning to unfold (9–12). The foundation for the function and selectivity of RGS proteins in regulating opioid signaling lies in their ability to interact with opioid receptors and their cognate G proteins. In general, the selectivity or the preference of an RGS protein for a particular receptor is determined by a variety of factors, including tissue-specific expression and precise interaction with the intracellular domains of receptor proteins, G protein subunits, and effectors as well as other pathway-specific components (13).The effects of RGS proteins on opioid receptor signaling have been examined in several systems. The findings are not always consistent, probably due to the different methodologies used. It has been shown that members of the RZ, R4, and R7 subfamilies (7) of RGS proteins play crucial roles not only in terminating acute opioid agonist action but also in opioid receptor desensitization, internalization, recycling, and degradation (3, 14), thereby affecting opioid tolerance and dependence (15–18). Much work has been performed with RGS4, because it is a smaller RGS protein with a structure consisting of the RGS consensus (box) sequence and a small N terminus (19, 20). It also has a wide distribution in the brain, especially in brain regions important for opioid actions, including the striatum, locus coeruleus, dorsal horn of the spinal cord, and cerebral cortex (21). In vitro RGS4 has been shown to reverse δ-opioid receptor agonist-induced inhibition of cAMP synthesis in membranes prepared from NG108-15 cells (6). Overexpression of RGS4 in HEK293 cells also attenuated morphine-, [d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin (DAMGO)-, and [d-Pen²,d-Pen⁵]enkephalin (DPDPE)-induced inhibition of adenylyl cyclase (22, 23). Co-expression of RGS4 with GIRK1/GIRK2 channels in Xenopus oocytes reduced the basal K⁺ current and accelerated the deactivation of GIRK channels activated by κ-opioid receptor agonist {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (24). Although these previous studies have provided evidence that RGS4 can negatively regulate opioid receptor signaling, they do not confirm a functional role for endogenous RGS4 in endogenous, nontransfected systems.Human neuroblastoma SH-SY5Y cells endogenously express μ- and δ-opioid receptors and a variety of Gα_i/o proteins (25–27). Here we show that RGS4 is abundantly found at both the mRNA and protein levels in these cells. Consequently, we used SH-SY5Y cells to examine the hypothesis that RGS4 negatively modulates opioid receptor signaling under physiological conditions. The endogenously expressed RGS4 level in SH-SY5Y cells was reduced using lentiviral delivery of short hairpin RNA (shRNA) targeting the RGS4 gene. This resulted in changes in δ- but not μ-opioid receptor-mediated signaling to adenylyl cyclase and the MAPK pathway. These findings argue for a selective interaction of RGS4 with the δ-opioid receptor. To test this, we expressed FLAG-tagged μ- and δ-opioid receptors together with a construct for a stable, proteosome-resistant RGS4 protein in HEK293T cells. Co-immunoprecipitation indicated that the δ-opioid but not the μ-opioid receptor was closely associated with RGS4, providing further evidence for a selective interaction between RGS4 and δ-opioid receptor signaling.     

Association of Nuclear Membrane Protein Lamin B1 with Necrosis and Apoptosis in Cell Death Induced by 5-Fluoro-2′-Deoxyuridine

We report that anticancer 5-fluoro-2 ′-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells was performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FUdR-treated original F28-7. Thus, the swelling feature for the necrosis was no longer observable, and instead cell shrinkage typical of apoptosis took place in almost all the cells examined. This finding suggests a new role for lamin B1 as a regulator in cell death.     

Different Mechanisms of Inhibition of DNA Synthesis by (E)-5-(2-Bromovinyl)-2′-deoxyuridine in Cells Transfected with Gene for Thymidine Kinase of Herpes Simplex Virus Type 1 and in Cells Infected with the Virus

Abstract

The effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) on deoxyribonucleoside 5′-triphosphate pools was studied in cells transfected with gene for thymidine kinase of herpes simplex virus type 1 and cells infected with the virus. When infected cells were treated with BVDU, the triphosphate form of the nucleoside analog was detected. When transfected cells were treated with BVDU, the triphosphate form was not detected and the pattern of changes in the pools was the same as after 5-fluoro-2′-deoxyuridine treatment. BVDU seems to inhibit DNA synthesis differently in the two cell lines and nucleotide metabolism in the transfected cells was not the same as in the infected cells.     

Determination of Concentration of Methotrexate Enantiomers in Intracellular and Extracellular Fluids of HepG2 Cells by Liquid Chromatography–Tandem Mass Spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG₂ cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1 % formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4 mL min^?1. The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 → m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL^?1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15 %. The mean recovery of (+)-MTX and (?)-MTX in the extracellular fluid of HepG₂ cells were 95.30 and 96.53 %, respectively, and the mean recovery of (+)-MTX and (?)-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12 %, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG₂ cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of (?)-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and (?)-MTX was (+)-MTX > (?)-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro.     

5-Bromovinyl 2′-Deoxyuridine Phosphorylation by Mitochondrial and Cytosolic Thymidine Kinase (TK2 and TK1) and Its Use in Selective Measurement of TK2 Activity in Crude Extracts

Mitochondrial thymidine kinase (TK2) is responsible for phosphorylation of thymidine and deoxycytidine and plays a crucial role in mitochondrial DNA precursor synthesis. TK2 is expressed in all tissues at low levels complicating accurate determinations, especially in tissues with high cytosolic thymidine kinase (TK1) activity. Recently, 5-bromovinyl 2 ′-deoxyuridine (BvdU) at 0.2 μ M was used to measure TK2 activity selectively. BvdU phosphorylation by pure human TK2 and TK1 was tested here, and the ratio of BvdU phosphorylation by TK2/TK1 was 91 at 0.2 μ M but was 500 at 2.5 μ M. Therefore, for reliable measurement of TK2 activity higher BvdU concentration should be used.     

Determination of genomic 5-hydroxymethyl-2’-deoxycytidine in human DNA by capillary electrophoresis with laser induced fluorescence

Recent studies reported the presence of 5-hydroxymethylcytosine (5 hmC) as an additional modification in mammalian genomic DNA. To date, 5 hmC has been detected only in mouse DNA isolated from embryonic stem cells, some adult tissues and in DNA from human bone marrow. Understanding its biological function will require the development of sensitive analytical methods that allow the detection and quantification of 5-hydroxymethylcytosine along with 5-methylcytosine and cytosine.

Here we report the validation of a fast and sensitive method for the quantification of global 5-hydroxymethyl-2'-deoxycytidine (5 hmdC) in DNA. The method is based on a procedure consisting of fluorescence labeling of deoxyribonucleotides and analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A double stranded DNA fragment containing a defined number of 5 hmdC residues was used for peak assignment, to establish separation conditions and to determine the limit of detection (LOD). The method yielded a LOD for 5 hmdC of 0.45 amol, which is equivalent to approximately to one 5 hmdC per 4,000 normal nucleotides (0.025%) using 1 μg of DNA as the matrix.

By applying the calibrated assay to the analysis of various DNAs we show that 5 hmdC is present in human tissue and human cancer cell lines. We demonstrate that by using CE-LIF DNA can be analyzed in one run for both methylation and hydroxymethylation of cytosine with high sensitivity and accuracy.     

Determination of α2HS-glycoprotein phenotypes by isoelectric focusing and immunoblotting: polymorphic occurrence of HSGA *5 in Okinawa

Summary A print immunofixation is a very useful procedure for the demonstration of ₂HS-glycoprotein (HSGA) following polyacrylamide gel isoelectric focusing. However, this technique has the one disadvantage of requiring a large volume of expensive antiserum. In this paper an alternative detection system is presented which involves the non-electric transfer of HSGA from a focused gel to a nitrocellulose filter and the immunologic detection of HSGA immobilized on nitrocellulose. Using this method the distribution of HSGA polymorphism in the Nepalese and Japanese populations was investigated. All the populations tested were found to lack the common HSGA ^*3 of the Caucasians. A rare HSGA ^*5 in Honshu, a main island of Japan, was observed at polymorphic frequency in Okinawa, Southern Japan.     

Protective Effects of Transforming Growth Factor β2 in Intestinal Epithelial Cells by Regulation of Proteins Associated with Stress and Endotoxin Responses

Transforming growth factor (TGF)-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC) in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs) remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS), and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.     

Telomere Shortening in Human HL60 Cells by Treatment with 3′-Azido-2′,3′-Dideoxynucleosides and Telomerase Inhibition by Their 5′-Triphosphates

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3′-azido-2′,3′-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.     

Visualization of NO3?/NO2? Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO₃⁻/NO₂⁻ exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO₃⁻/NO₂⁻ for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology), by employing NO₃⁻/NO₂⁻-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO₃⁻ and NO₂⁻, and its FRET signal is changed in response to NO₃⁻/NO₂⁻ in the micromolar range. Furthermore, both an increase and decrease in cellular NO₃⁻ concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO₃⁻/NO₂⁻ dynamics in various organisms, including plants and animals.