Localization by in situ hybridization of steroid 5alpha-reductase isozyme gene expression in the human prostate and preputial skin

PURPOSE: The synthesis of dihydrotestosterone is catalyzed by steroid 5alpha-reductase isozymes, designated as types 1 and 2. Controversial results have been reported on the identification of the cell types expressing each isozyme in the human prostate and genital skin. The objective of the present study was to clearly identify at the cellular level the cells containing each type of 5alpha-reductase isozyme. MATERIALS AND METHODS: We used for the first time an in situ hybridization technique involving use of [35S]-labeled oligonucleotide probes to determine the cell type expression patterns of the two 5alpha-reductase isozymes in human prostate and preputial skin. RESULTS: In the prostate, hybridization with types 1 and 2 5alpha-reductase antisense probes led to a positive reaction in both epithelial and stromal cells, while the sense probes did not generate any signal. The silver grain counts for both isozymes revealed a higher labeling in the epithelial cells. In fact, the ratio epithelial cells/stroma was around 2 for both 5alpha-reductase types 1 and 2. In the preputial skin, 5alpha-reductase type 1 was found to be highly expressed in all the layers of the epidermis with the exception of the stratum corneum while a lower labeling was observed in some fibroblasts as well as in the secretory cells of sebaceous glands and excretory duct cells of sweat glands. Type 2 5alpha-reductase showed a very similar pattern of distribution. CONCLUSION: It appears that, in two human tissues, the two 5alpha-reductase isozymes mRNAs are expressed by the same cell types. This new observation should help clarify the physiological role of each type of 5alpha-reductase isozyme.     

Prenatal exclusion of segmental trisomy in familial chromosome 21 pericentric inversion by fluorescence in situ hybridization

We report the prenatal exclusion of partial trisomy in a family with maternal pericentric inversion of chromosome 21 by fluorescence in situ hybridization (FISH). After determining the structural rearrangement in the mother and her affected son with 46,XY,rec(21)dup(21q)inv(21)(p11q22) resulting in Down syndrome (DS), a chorionic villus sample from the current pregnancy was analysed for the copy number of the DS critical region with a cosmid contig. The signal distribution was normal and the cytogenetic analysis revealed that the fetus had inherited the inverted chromosome 21 in a balanced form. FISH probes specific for the DS region are of great value in supporting cytogenetic results, regardless of the structural status of chromosome 21.     

Non-isotopic in situ hybridization to detect chick Sox gene mRNA in plastic-embedded tissue

OBJECTIVES: Evaluate the ability of two bone alkaline phosphatase (ALPB) immunoassays (Ostase, Hybritech Inc and Alkphase-B, Metra Biosystems) to clinically differentiate between osseous and non-osseous ALP sources. DESIGN AND METHODS: Specimens from patients with either liver or bone disease (Paget's disease or metastatic cancer) were analyzed by both methods. RESULTS: There was a good correlation between these two assays. Values for ALPB, whether determined as a concentration by the Ostase assay or as an activity by the Alkphase-B assay, were similar for subjects with liver disease or bone disease. However, total ALP (ALPT) activity was higher in liver disease compared to bone. When ALPB was expressed in relation to ALPT, ratios were significantly greater in subjects with bone disease than in those with liver disease. ALPB/ALPT ratios improved the specificity of the Ostase assay from 52% to 86% and the Alkphase-B assay from 58% to 74%. CONCLUSIONS: These two ALPB assays have good analytical performance and their clinical utility can be enhanced by expressing ALPB values in relation to ALPT activity.     

Advances in fluorescence in situ hybridization

The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates

The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 (HSP90 alpha) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90 alpha were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90 alpha. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90 alpha and the C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.     

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.     

Semiautomatic image analysis for grain counting in in situ hybridization experiments

We have developed a computer image analysis procedure for counting autoradiographic grains in in situ hybridization experiments. The procedure automatically estimates the number of autoradiographic grains over cells and measures cell number and size so that grain density per unit cell area can be calculated. Advantages include the clear separation of grains and cells, using chromatic and spatial filters to enhance the image; the use of gray level operators to extract cells from grains; and the use of binary operators for separating apposed or partially overlapping cells and grains. Comparison of manual and automated grain counts revealed a significant correlation between human and computer estimations of grain number. However, the automatic grain counting technique consistently underestimated the number of grains when grain density was high. Measures of the fractional area occupied by grains normalized by the average area of a single grain were a better estimate at high grain densities. The procedure can be modified easily to operate on most image analyzers.     

Use of photo-anchoring of DNA probes for fluorescent in situ hybridization

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.     

Detection of the leghemoglobin gene on two chromosomes of Phaseolus vulgaris by in situ PCR linked-fluorescent in situ hybridization (FISH)

OBJECTIVE: To investigate the effect of phentolamine, a sympathetic blocking agent, on the spontaneous electrical activity (SEA) recorded from a locus of a myofascial trigger spot (MTrS), equivalent to a human trigger point, in rabbit skeletal muscle. DESIGN: Randomized control trial. SETTING: A university medical laboratory. PATIENTS OR OTHER PARTICIPANTS: Nine adult New Zealand rabbits. INTERVENTION: In the experimental group phentolamine mesylate (1mg/kg) was injected into the external iliac artery, followed by flushing with normal saline. The control group was treated with normal saline instead of phentolamine using the same procedure. MAIN OUTCOME MEASURES: SEA was recorded from multiple active loci of MTrSs in the biceps femoris muscle: initially SEA in the same locus was recorded before and immediately after phentolamine (or normal saline) injection; then SEA was recorded from 25 different active loci. The mean of the average integrated signal (AIS) of SEA was analyzed, comparing the effects of phentolamine and normal saline on SEA. RESULTS: In the same active locus, the AIS of SEA showed statistically a linear decay with time after phentolamine injection, with a correlation coefficient of .56 at p < .05. However, no statistical relationship could be derived for the control group data with time by using regression analysis, probably because of large variations among the rabbits and movement artifacts during the experiment. In 25 different loci in the phentolamine group, the mean of the AIS of SEA (7.92 microV) was significantly lower than that of the control group (9.89 microV) at p < .05. CONCLUSIONS: The results support the hypothesis that the autonomic nervous system is involved in the pathogenesis of myofascial trigger points. The application of the AIS as an evaluation index seems to be feasible in the quantitative measurement of SEA.     

Efficacy of fluorescence in situ hybridization for detecting PML/RARA gene fusion in treated and untreated acute promyelocytic leukemia

OBJECTIVE: To test the efficacy of fluorescence in situ hybridization (FISH) for detection of fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes in patients with treated or untreated acute promyelocytic leukemia (APL). DESIGN: We conducted a retrospective blind study on a series of stored bone marrow specimens from normal subjects and patients with APL. MATERIAL AND METHODS: Conventional cytogenetic and FISH analyses were done on interphase and metaphase cells in specimens from 31 normal subjects and 19 patients with untreated or treated APL. RESULTS: From 25 of the normal specimens, we calculated a normal cutoff of 10% for interphase cells and 0% for metaphase cells. With use of these criteria, the other six specimens from normal subjects showed normal findings, and each of the seven specimens from patients with untreated APL was abnormal by FISH analysis. The specimens from four patients in clinical relapse or with residual APL were abnormal. Of the eight specimens from patients in clinical remission, three were abnormal; two of these patients had a relapse within 8 months, and the other patient had received 1 month of chemotherapy and was entering remission. Of the other five patients in remission, four had normal FISH results and have now been in remission for 2.5 to 10 years. The other patient in remission with normal FISH results had a relapse within 6 months. PML/RARA fusion was detectable in three patients with hypogranular APL and in three with a cytogenetic variant of the t(15;17). CONCLUSION: The results of this study suggest that FISH with PML and RARA probes can be used to diagnose APL and may be useful for monitoring treated patients.     

Localization of PTH gene on pig chromosome 14q25-q28 by in situ hybridization

BACKGROUND AND OBJECTIVES: A survey of anesthesia practice was conducted among French residents in anesthesia at the end of their training. This study was performed mainly to evaluate the residents' experience in peripheral nerve blocks. METHODS: Two short clinical cases were proposed to all French residents during a telephone interview immediately before their certification. The first described the case of a young asthmatic patient admitted for an elbow fracture. The second described an elderly woman with severe aortic stenosis admitted for a supracondylar fracture of the femur. A questionnaire had been prepared and was filled in during the interview. Each resident was asked to answer according to the actual choice he or she would have made. For both cases, when general anesthesia was chosen first, the next question was to discuss which regional anesthesia would be used if general anesthesia had to be discarded. In that way, the practical knowledge about most common peripheral nerve blocks learned during residency was investigated. RESULTS: Of 77 residents registered as being at the end of their residency, 8 were on either sabbatical or maternity leave. Regional anesthesia was the first choice in 78% and 57% of cases for the first and second clinical cases, respectively. The regional anesthetic techniques chosen were axillary block (66%), interscalene block (31%), and intravenous regional anesthesia (3%) for case 1 and combined lumbar plexus and sciatic block (36%), epidural anesthesia (30%), single-shot spinal anesthesia (18%), and continuous spinal anesthesia (16%) for case 2. Throughout the residency of the group, 32 +/- 2 axillary blocks, 12 +/- 2 interscalene blocks (axillary vs interscalene, P < .0001), 21 +/- 3 femoral blocks, and 10 +/- 2 sciatic blocks (femoral vs sciatic, P < .0001) had been performed (mean +/- SEM). They had also performed 2.5 +/- 0.5 continuous spinal anesthesias and 17 +/- 3 intravenous regional anesthesias respectively. Upper extremity blocks were more often used during residency than lower extremity blocks (44 +/- 3 vs 31 +/- 4, P < .01). A peripheral nerve stimulator was routinely used by 83% of residents. CONCLUSION: French residents in anesthesiology at time of certification are better trained for peripheral nerve blocks of the upper extremity than for those of the lower extremity. Axillary plexus and femoral nerve block are the most widely used blocks, probably reflecting the techniques the most mastered among teachers. Finally, the extensive use of a peripheral nerve stimulator by residents is probably the result of the widespread use of this device by teachers in France.     

Localization of AT2 angiotensin II receptor gene expression in rat brain by in situ hybridization histochemistry

To localize the gene expression of AT2 angiotensin II receptors in rat brain we performed in situ hybridization histochemistry using 35S-labeled antisense riboprobes. The AT2 receptor mRNA expression pattern was compared in consecutive brain sections, from 2 week old rats, with the receptor expression by means of [125I]Sar1-ANG II binding and displacement with AT2 selective ligands followed by autoradiography. Expression of AT2 receptor mRNA was found in several thalamic nuclei (ventral posterolateral, mediodorsal, central medial, paracentral, and paraventricular), the medial geniculate nuclei, the nucleus of the optic tract, the subthalamic nucleus, the interposed nucleus of the cerebellum, and in the inferior olive. In these areas the AT2 receptor gene expression corresponds well with [125I]Sar1-ANG II binding. In addition, AT2 receptor mRNA expression was found in the red nucleus where no [125I]Sar1-ANG II binding was present. No significant hybridization of the AT2 receptor antisense probe was found in septal nuclei, the locus coeruleus, the dorsolateral geniculate nucleus, or the cerebellar cortex, areas rich in [125I]Sar1-ANG II binding. Our results indicate that some brain regions may be involved in AT2 receptor formation, transporting the receptor protein to other brain areas. However, in most structures, both the formation and expression of receptors occur, suggesting the existence of local AT2 receptor circuits, or that of AT2 autoreceptors. Other structures express only the receptor protein, indicating that these AT2 receptors are produced elsewhere. Our present data are the basis for further studies on the clarification of AT2 receptor pathways in the brain.     

Cloning and characterization of the cDNA for the murine iduronate sulfatase gene

Iduronate sulfatase (IDS; EC 3.1.6.13) is a lysosomal enzyme that acts on sulfate groups on C-2 positions of iduronic acid residues of the mucopolysaccharides dermatan and heparan sulfate. A deficiency of this enzyme activity in man leads to Hunter syndrome (Mucopolysaccharidosis type II). We report here the cloning and sequence characterization of the murine iduronate sulfatase cDNA which encodes 564 amino acid residues. Within the coding region the murine gene is 84.9 and 84.5 identical to the human gene at the nucleotide and amino acid levels, respectively. The two regions containing the putative catalytic site are especially well conserved. Genetic mapping of the murine Ids cDNA in an interspecific backcross confirms an X chromosomal location between Fmr-1 and Gabra3.