Detection of receptor mRNAs using nonradioactive in situ hybridization

To understand better the regulatory mechanism of cell function through bioactive substance such as hormones and cytokines, analysis of the gene expression of their receptors at the individual cell level is required. Since the presence of specific mRNA is a good index of gene expression, in situ hybridization (ISH) has been recognized as a powerful tool. Especially, nonradioactive ISH with synthetic oligodeoxynucleotide probe is a safe, quick and highly sensitive method. We have developed a unique method with thymine-thymine (T-T) dimer as a nonradioactive labeling, in which adjacent thymines are dimerized by ultraviolet light irradiation. The T-T dimers in hybrids are immunohistochemically recognized using an anti-T-T dimer antibody. In this article, we will present an example of ISH protocol, which works very well both T-T dimer and digoxigenin-labeled probes.     

Direct assessment of triploid cells in mosaic human fetuses by fluorescence in-situ hybridization

Villous tissues from 30 spontaneous abortions and the same number of artificial abortions were obtained and analysed for the frequency of polyploid cells. Single cell suspensions were made from these tissues without culture and the ploidy of > 100 cells was analysed. Trisomies of chromosomes 17 and 4 have rarely been reported in villous cells of spontaneous abortions, suggesting that the presence of more than three copies of chromosomes 17 and 4 per cell indicates polyploidy. The number of chromosomes 17 and 4 was detected by fluorescence in-situ hybridization analysis using centromeric probes D17Z1 and D4Z1. Most villous cells from cases of spontaneous and artificial abortions had two D17Z1 or D4Z1 signals per cell, with very small percentages of cells (0.5 +/- 0.4%) showing three signals per cell. However, in four cases of spontaneous abortions, 2-12% of cells had three D17Z1 or D4Z1 signals per cell. This indicates the presence of triploid cells in these cases of spontaneous abortion, at a significantly higher frequency compared to artificial or the remaining 26 cases of spontaneous abortion. In addition, three cases contained 0.2-0.4% of cells showing six signals, indicating that these cells were dividing triploid cells. The low frequency of mosaicism reported here would not be detectable by conventional chromosomal analysis.     

Quantitative in situ hybridization of three gonadotropin-releasing hormone-encoding mRNAs in castrated and progesterone-treated male tilapia

We investigated the effects of castration and progesterone administration on the three gonadotropin-releasing hormone (GnRH)-encoding mRNAs in sexually mature male tilapia Oreochromis niloticus. In situ hybridization histochemistry was performed using 35S-labeled antisense oligonucleotide probes complementary to salmon-, seabream-, and chicken II-GnRH cDNAs to quantify cellular GnRH mRNA expression in the terminal nerve ganglia (nucleus olfactoretinalis), preoptic area, and midbrain tegmentum of animals castrated for 2 weeks and injected intraperitoneally with sesame oil or progesterone. Castration significantly elevated salmon-GnRH mRNA but not seabream- or chicken II-GnRH mRNA levels. Progesterone treatment had no effect on salmon-, seabream-, or chicken II-GnRH mRNA levels. Comparisons between intact, castrated, and progesterone-treated animals showed no change in the total volume of nucleus olfactoretinalis, cell sizes, and total numbers of cells expressing GnRH mRNA within the midbrain and preoptic area. These results demonstrate that salmon-GnRH but not seabream- or chicken II-GnRH-synthesizing neurons are under a gonadal steroid negative feedback control and that progesterone might not be the main hormone regulating the three GnRH-encoding mRNAs in the male tilapia.     

A system for the quantitation of DNA using a microtiter plate-based hybridization and enzyme amplification technology

A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P1. The pH-dependent specificity of this enzyme for 3'-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Direct quantitation of RNA transcripts by competitive single-tube RT-PCR and capillary electrophoresis

BACKGROUND: Patients with congestive heart failure (CHF) have a reduced exercise capacity because of the early appearance of fatigue and dyspnea. Qualitative changes in the skeletal muscle composition and metabolism can be responsible for the origin of symptoms METHODS: We correlated the myosin heavy chain (MHC) composition of the gastrocnemius in 20 patients with different degrees of CHF to NYHA class, diuretic consumption, echocardiographic parameters, and expiratory gases measured during cardiopulmonary exercise testing. MHC composition was determined electrophoretically in skeletal muscle needle microbiopsies and the percent distribution was calculated by densitometry. Maximal cardiopulmonary exercise testing was performed on a treadmill with a modified Naughton protocol. A capnograph was used. RESULTS: There was no correlation between ejection fraction, left ventricular end systolic diameter, left ventricular end diastolic diameter, and MHC composition. We found a significant positive correlation between the percentage of MHC 1 (slow aerobic isoform) and NYHA class (r2 = 0.62, p < 0.0001), peak VO2 (r2 = 0.5, p < 0.0004), ventilatory threshold (VT) (r2 = 0.33, p = 0.008) and O2 pulse (peak VO2/HR) (r2 = 0.40, p = 0.003). There was a negative correlation between both MHC2a (fast oxidative) and MHC2b (fast glycolytic) with peak VO2 (r2 = 0.38, p = 0.004 and r2 = 0.37, p = 0.004, respectively), VT (r2 = 0.2, p = 0.046 and r2 = 0.34, p = 0.007, respectively), and O2 pulse (peak VO2/HR) (r2 = 0.39, p = 0.003 and r2 = 0.23, p = 0.03). NYHA class was also correlated positively with MHC2a and MHC2b (r2 = 0.46, p = 0.001 and r2 = 0.41, p < 0.006, respectively) and negatively with the same clinical and functional parameters. CONCLUSIONS: The correlation between the magnitude of the MHC shift from the slow aerobic to the fast glycolytic and fast oxidative with both functional and objective measurements of exercise capacity (peak VO2, VT, O2 pulse) seem to suggest that changes in skeletal muscle composition may play a determining role in exercise tolerance in patients with CHF.     

Immunochemical visualization and quantitation of cyclic AMP in single cells

Adenosine 3':5'-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.     

Semi-quantitative RT-PCR for comparison of mRNAs in cells with different amounts of housekeeping gene transcripts

There is a need for more information about research utilization and other related research activities among nurses in the U.K. This article reports on data from a large scale survey of nurses in Northern Ireland, in particular their self-reported extent of research utilization, their attitudes to research, research projects carried out and their frequency of accessing research information. A questionnaire was administered to a convenient sample of 2600 nurses from 23 hospitals in 14 Trusts. The number of responses was 1368 (52.6%). The findings show that although respondents held positive attitudes to research, only about a third reported utilizing research 'frequently/all the time' and less than 40% reported that they had implemented research findings in the two years prior to the survey. These figures were, however, higher for degree nurses. The implications of these and other findings are discussed.     

Direct measurement of single-molecule diffusion and photodecomposition in free solution

Continuous monitoring of submillisecond free-solution dynamics of individual rhodamine-6G molecules and 30-base single-stranded DNA tagged with rhodamine was achieved. Fluorescence images were recorded from the same set of isolated molecules excited either through the evanescent field at the quartz-liquid interface or as a thin layer of solution defined by micron-sized wires, giving diffraction-limited resolution of inter-connected attoliter volume elements. The single-molecule diffusion coefficients were smaller and the unimolecular photodecomposition lifetimes were longer for the dye-DNA covalent complex as compared with those of the dye molecule itself. Unlike bulk studies, stochastic behavior was found for individual molecules of each type, and smaller diffusion coefficients were observed.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.     

原子吸收法直接测定硫代硫酸铵溶液浸金系统中金的研究

硫代硫酸铵溶液中金可用原子吸收法直接测定。方法相对标准偏差为２０％。浸取过程中Ｃｕ＾２＋，ＮＨ４ＯＨ，（ＮＨ４）２Ｓ２Ｏ３，（ＮＨ４）２ＳＯ４等浓度对测定无影响。方法简单、易操作。取样体积小。     

Distribution and expression of mRNAs for the proto-oncogenes c-fos and c-jun in bone cells in vivo

Aminopeptidase A is a homodimeric membrane-bound zinc metallopeptidase anchored at the plasma membrane by a 22-amino-acid hydrophobic segment. The anchor segment separates a small N-terminal cytoplasmic domain from a large ectodomain that contains the active site. Site-directed mutagenesis was performed to investigate the role of the cytoplasmic domain of aminopeptidase A in membrane anchoring and routing of the enzyme. Expression in COS-7 cells of a mutant lacking the N-terminal cytoplasmic domain resulted in the efficient secretion of a catalytically active enzyme in the medium. The soluble mutated aminopeptidase A, purified from the medium of a stable cell line, exhibited similar biochemical features to those of the wild-type enzyme. Pulse/chase metabolic labeling experiments revealed that the soluble form is generated intracellularly at an early stage of biosynthesis, suggesting that the signal peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.     

Direct detection of Mycobacterium tuberculosis in sputum by polymerase chain reaction and DNA hybridization

A polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary tuberculosis was developed by using oligonucleotide primers to amplify a fragment of IS6110, an insertion sequence repeated multiple times in the chromosome of Mycobacterium tuberculosis. Sediment obtained from sputa processed by the N-acetyl-L-cysteine-NaOH method was suspended in a simple lysis buffer and was heated at 100 degrees C for 30 min prior to amplification. A dUTP-uracil N-glycosylase PCR protocol was used to prevent false-positive test results because of the carryover of products from previous amplification reactions. The 317-bp amplicon was detected by direct gel analysis and Southern blotting and then hybridization with a biotin-labeled internal probe. Hybrid molecules were detected by using a commercially available avidin-alkaline phosphatase-chemiluminescent substrate system (Tropix, Inc., Bedford, Mass.). The analytical sensitivity of the assay was 10 fg of purified mycobacterial DNA. The limits of detection by culture (Middlebrook 7H11 agar and Lowenstein-Jensen medium) and by PCR were equivalent in terminal dilution experiments for organism suspensions and positive sputa. An internal control was used to detect the presence of amplification inhibitors in each negative reaction mixture. DNA was purified from inhibitory specimens by phenol-chloroform extraction and ethanol precipitation. PCR results were compared with results of microscopy and conventional culture for the detection of M. tuberculosis in 313 sputum specimens. There were 124 specimens that were positive for M. tuberculosis by conventional methods and 113 (91%) that were positive by PCR. PCR detected 105 of 110 (95%) of the smear-positive and 8 of 14 (57%) of the smear-negative specimens. There were no false-positive results by PCR (specificity, 100%). This PCR assay innovations that make application of this new technology feasible in clinical microbiology laboratories.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

Comparison between catalytic activity and activity changes during denaturation by guanidine hydrochloride of enzymes in crystalline state and in solution

The catalytic activity and activity changes during denaturation by guanidine hydrochloride of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and alpha-chymotrypsin in crystalline state and in solution have been compared. The catalytic activities are lower in crystalline state than in solution. Enzymes in crystalline state are more stable than in solution during denaturation by guanidine hydrochloride. Ammonium sulfate has different effects on catalytic activities of different enzymes and shows protection on all enzymes studied during denaturation by guanidine hydrochloride. The protection is more obvious at high concentrations of guanidine hydrochloride than at low concentrations. It is suggested that the flexibility or mobility of enzyme is required for the catalytic activity and related to the stability of enzymes. Enzymes with less flexibility or mobility are more stable.