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Biomedical subjects

R Sanchez

Publications and source records attributed to R Sanchez.

At least 91 records · Page 5Linked to original sources
PubMed1995

Structure-function relationships and molecular genetics of the 3 beta-hydroxysteroid dehydrogenase gene family.

The isoenzymes of the 3 beta-hydroxysteroid dehydrogenase/5-ene-4-ene-isomerase (3 beta-HSD) gene family catalyse the transformation of all 5-ene-3 beta-hydroxysteroids into the corresponding 4-ene-3-keto-steroids and are responsible for the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. The two human 3 beta-HSD genes and the three related pseudogenes are located on the chromosome 1p13.1 region, close to the centromeric marker D1Z5. The 3 beta-HSD isoenzymes prefer NAD+ to NADP+ as cofactor with the exception of the rat liver type III and mouse kidney type IV, which both prefer NADPH as cofactor for their specific 3-ketosteroid reductase activity due to the presence of Tyr36 in the rat type III and of Phe36 in mouse type IV enzymes instead of Asp36 found in other 3 beta-HSD isoenzymes. The rat types I and IV, bovine and guinea pig 3 beta-HSD proteins possess an intrinsic 17 beta-HSD activity specific to 5 alpha-androstane 17 beta-ol steroids, thus suggesting that such "secondary" activity is specifically responsible for controlling the bioavailability of the active androgen DHT. To elucidate the molecular basis of classical form of 3 beta-HSD deficiency, the structures of the types I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients were analyzed. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, at the exception of L108W and P186L proteins, which have some residual activity (approximately 1%). Mutations found in nonsalt-loser patients have some residual activity ranging from approximately 1 to approximately 10% compared to the wild-type enzyme. Characterization of mutant proteins provides unique information on the structure-function relationships of the 3 beta-HSD superfamily.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Assessment of vitamin and trace element supplementation in severely burned patients undergoing long-term parenteral and enteral nutrition.

The efficacy of an oral supplement of vitamins and trace elements during a longterm artificial parenteral and enteral nutrition was investigated for 3 months in patients with extensive burns. Thirty severely burned patients (22 male, 8 female, age 41 +/- 18 years, range 23-59 years, 33 +/- 12% total body surface area burn, 22% +/- 8 full thickness burn surface area) were included. Every 10 days, from day 10 until day 90, we determined serum levels of: *vitamins B1, B12, A, E, *folic acid, *copper, zinc, iron, *transferrin, albumin, prealbumin, total proteins, *fibronectin, retinol binding protein (RBP), *calcium, *phosphorus, *triglycerides, *total cholesterol, *C reactive protein (CRP), *erythrocyte folic acid. The mean daily nutritional support was 60 Kcals and 0.4 g N per kg of body weight, 70% enterally and 30% parenterally administered, with enteral vitamin and trace element supplementation. On day 10, there was a decrease of the serum level of 19/20 parameters. For 8 parameters (vitamin A, total cholesterol, iron, transferrin, fibronectin, phosphorus, RBP, total proteins), the level was lower than usual. Between day 10 and day 20, a significant normalization of 6 of them was noted, the average levels of transferrin and iron remaining below normal values until day 50. There was a significant decrease in C-reactive protein levels, however above normal limits. No deficiency in vitamins or trace elements was found. Cyclic variations of serum levels occurred which may be more related to volemic, hydroelectrolytic, endocrine and inflammatory disorders than to nutritional problems.

Journal Article↗
PubMed1995

Nonsalt-losing male pseudohermaphroditism due to the novel homozygous N100S mutation in the type II 3 beta-hydroxysteroid dehydrogenase gene.

Recently, the structure of two genes encoding isoenzymes responsible for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) activity in the human was elucidated. This activity is an essential step in the biosynthesis of all classes of steroid hormones. In the classic severe form of 3 beta HSD deficiency, patients present with adrenal insufficiency, various degrees of salt loss, and incomplete masculinization in males. Here we report the characterization of the molecular basis of congenital adrenal hyperplasia due to 3 beta HSD deficiency in a male pseudohermaphrodite born from consanguineous parents and having no clinical salt loss. To analyze the structure of the type I and II 3 beta HSD genes of the patient, DNA fragments, generated by polymerase chain reaction amplification of the four exons and the exon-intron boundaries of these genes, were directly sequenced. The patients carried a homozygous missense mutation converting Asn100 to Ser in exon 3 of his type II 3 beta HSD gene. His parents were heterozygous for the same point mutation. The absence of clinical salt loss associated with a male pseudohermaphroditism suggested that 3 beta HSD activity was impaired to different levels in the testes and adrenal. To elucidate whether this N100S missense mutation affected preferentially a steroidogenic pathway, enzymatic activity was analyzed by in vitro analysis of mutant recombinant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 cells. Using homogenates from transfected cells, the N100S 3 beta HSD enzyme showed a Km value for pregnenolone of 25 +/- 3 mumol/L compared with 3.5 +/- 0.2 mumol/L for the normal human type II 3 beta HSD enzyme. Similar results were obtained using dehydroepiandrosterone as substrate. In addition to decreasing apparent affinity, the N100S mutation decreased the relative specific activity (Vmax), leading to a relative specificity (relative Vmax/Km) 2.7% and 11% that of normal type II 3 beta HSD using pregnenolone or dehydroepiandrosterone as substrate, respectively. Moreover, the mutant N100S protein had an apparent decreased affinity for NAD+, with a Km value of 650 +/- 66 mumol/L compared with 20 +/- 2 mumol/L for normal type II 3 beta HSD. Except for the hypothetical effect of local factors, these findings suggest that a very weak residual activity of the normal type II 3 beta HSD enzyme could prevent salt loss, but it was insufficient for normal male sex differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

A serological study on infection patterns, control and persistence of classical swine fever in infected farms in the Philippines.

In 3 farrow-to-finish farms with enzootic classical swine fever (CSF) all sick pigs were culled and an intensive vaccination program was introduced. Boars and cows were vaccinated every 6 months and piglets at the age of 6 and 8 weeks. The infection was monitored on clinical grounds and by means of serological examination of the farms. Serological results reflected well the clinical situation in the farms. High titers and high percentage of animals with positive titers were observed in infected farms, compared to lower titers in a clinically "free" farm, which were interpreted as titers due to vaccination. Although clinical improvement was noticed, the rigid vaccination regime was not able to control CSF in none of the farms. In all cases, a small percentage of pigs in the finishing stage was still affected. Since these pigs seemed not to have responded to vaccination at 6 and 8 weeks of age, the programme was questioned. Additionally, very high titers were recorded in sows from infected farms, which would automatically extend the period of passive protection in offspring and postpone the age at which piglets could be vaccinated. More intensive programmes, which include also vaccination beyond the age of 8 weeks, were suggested to bring enzootic infections under control.

Aging↗
PubMed1995

Development of human papillomavirus type 16 associated squamous cell carcinoma of the scrotum in a patient with Darier's disease treated with systemic isotretinoin.

In contrast to squamous cell carcinoma of the penis, scrotal carcinoma has historically been associated with exposure to environmental or industrial carcinogens and has only rarely been correlated with human papillomavirus. We report on a patient with squamous cell carcinoma of the scrotum in which human papillomavirus type 16 was integrated into the tumor cell genome, suggesting a causal role of human papillomavirus in the development of squamous cell carcinoma of the scrotum. Other unique features of our case include the presence of Darier's disease, an uncommon genodermatosis, and treatment with oral retinoids, which have prophylactic value in the prevention of cutaneous malignancies.

Amino Acid Sequence↗
PubMed1994

NMDA receptor-mediated synaptic input to nitric oxide synthase-containing neurons of the guinea pig mesopontine tegmentum in vitro.

Intracellular recordings were obtained from laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) neurons in a slice preparation to determine if excitatory synaptic input to the nitric oxide synthase (NOS)-containing cells was mediated by NMDA receptors. NOS cells were identified by intracellular injection of biocytin and NADPH-diaphorase histochemistry. Application of NMDA produced membrane depolarization, and EPSPs were reversibly reduced by an average of 46.3 +/- 16.6% with AP-5 (50 microM). These results indicate that post-synaptic NMDA receptors participate in synaptic transmission to NOS-containing neurons in LDT and PPT and are consistent with the hypothesis that synaptic NMDA receptors may control NO production in these cells.

2-Amino-5-phosphonovalerate↗
PubMed1994

Temporal relationship of complications after coronary artery bypass graft surgery: scheduling for safe discharge.

The postoperative courses of 224 patients who left the operating room alive after coronary bypass surgery were followed to determine the time of onset of complications to determine when discharge is safe. A total of 155 complications occurred in 103 patients including 59 with supraventricular tachycardia, 17 with fever, and 9 to 11 with ventricular arrhythmias, cerebrovascular accidents, wound infection, or deep vein thrombosis. Pneumonia occurred in seven patients, and other complications occurred in five or fewer patients. Most initial complications (77.6%) occurred by day 5, and 89.3% of the patients with complications had their initial event before day 8. Hence it appears that it is safe to discharge patients on day 8 after coronary artery bypass graft surgery if they have not had a postoperative complication.

Aged↗
PubMed1994

Formation and degradation of dihydrotestosterone by recombinant members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase family.

The structures of cDNA clones encoding four members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family were characterized. The rat type I, type II and the novel type IV are genuine NAD+/H-dependent 3 beta-HSD isoenzymes. On the other hand, the liver-specific type III protein is a specific 3-keto-reductase (3-KSR) that catalyzes the conversion of 5 alpha-androstane-3-one-17 beta-ol (DHT) and 5 alpha-androstane 3,17-dione (A-dione) into their 3 beta-hydroxy metabolites. The aim of the present study was to further characterize the enzymatic properties of rat types I, III and IV, especially their role in the formation and degradation of DHT after transient expression in intact human HeLa cervical carcinoma, JEG-3 choriocarcinoma or SW-13 adrenal cortex adenocarcinoma cells in culture. The expressed type III 3-KSR in intact HeLa cells catalyzed the reduction of DHT into 3 beta-diol, whereas expression of type I 3 beta-HSD in these cell lines had no significant effect on the basal conversion of DHT into 3 beta-diol, but it did increase the formation of DHT from 3 beta-diol. A-dione is the predominant product obtained when DHT and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) are used as substrates in intact JEG-3 and SW-13 cells transfected with rat type I 3 beta-HSD. Furthermore, this predominant 17 beta-HSD activity was also observed in SW-13 cells transfected with the novel rat type IV 3 beta-HSD. The predominance of this 'secondary' 17 beta-HSD activity is also reflected in HeLa cells transfected with type I 3 beta-HSD by the deduced predominant pathway 3 beta-diol-->DHT-->5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol)-->androsterone (ADT), in which formation of 3 alpha-HSD activity of HeLa cells, whereas the other reactions are catalyzed by the type I 3 beta-HSD isoenzyme. This observation thus demonstrates that rat type I 3 beta-HSD may also catalyze the conversion of 3 alpha-diol into ADT through its intrinsic 17 beta-HSD activity. The predominant metabolic pathways observed in the present study could be attributed to preponderant bioavailability of NAD+ and NADPH in the intact transfected cells used.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Functional characterization of the novel L108W and P186L mutations detected in the type II 3 beta-hydroxysteroid dehydrogenase gene of a male pseudohermaphrodite with congenital adrenal hyperplasia.

Two isoenzymes are responsible for 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) activity in humans. We analyzed the structure of types I and II 3 beta-HSD genes in a male pseudohermaphrodite suffering from a severe salt-losing form of congenital adrenal hyperplasia. We did not detect any mutation in the type I 3 beta-HSD gene, but we found two different missense mutations in exon IV of the type II 3 beta-HSD gene of the patient; a conversion of codon Leu108 into a Trp (L108W) inherited from his mother and a conversion of codon Pro186 into a Leu (P186L) inherited from his father. We assessed the effect of the L108W and P186L mutations on 3 beta-HSD activity by in vitro analysis of mutant enzymes expressed in heterologous COS-1 cells. Using homogenates from transfected cells, the Km values for PREG were 7 +/- 2 and 8 +/- 2 microM for the recombinant L108W and P186L enzymes, respectively, compared with 2.2 +/- 0.2 microM for the normal type II 3 beta-HSD enzyme. Moreover, Km values for NAD+ were much higher for the L108W and P186L proteins, being 678 +/- 166 and 920 +/- 351 microM, respectively, compared with 24 +/- 3 microM for the normal type II 3 beta-HSD enzyme. Vmax values for PREG and NAD+ were lower for both mutant enzymes; thus, the in vitro overall efficiency, relative to the normal enzyme, is approximate as 0.3% and 0.2% for the L108W and P186L enzymes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Detection and functional characterization of the novel missense mutation Y254D in type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene of a female patient with nonsalt-losing 3 beta HSD deficiency.

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) deficiency is a form of congenital adrenal hyperplasia characterized by severe impairment of steroid biosynthesis in the adrenals and gonads. To better understand the molecular basis of the phenotypic heterogeneity found in 3 beta HSD deficiency, we analyzed the structure of type I and II 3 beta HSD genes in a female patient with nonsalt-losing 3 beta HSD deficiency diagnosed at puberty. We directly sequenced DNA fragments generated by polymerase chain reaction amplification of the four exons, the exon-intron boundaries, and the 5'-flanking regions of each gene. No mutation was detected in the type I 3 beta HSD gene, which is the predominant species expressed in the placenta and peripheral tissues. We detected a novel missense mutation, Y254D, in one allele of the patient's type II 3 beta HSD gene, which is the almost exclusive type expressed in the adrenals and gonads. The influence of the Y254D mutation on enzymatic activity was assessed by analyzing the recombinant mutant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 monkey kidney cells. Recombinant mutant type II 3 beta HSD enzyme carrying the Y254D substitution exhibits no detectable activity with C21 delta 5-steroid pregnenolone or C19 delta 5-steroid dehydroepiandrosterone used as substrate. The absence of restriction fragment length polymorphism by Southern blot analysis and the finding that all of the amplified DNA fragments possess the expected length suggest the absence of deletions, duplications, or re-arrangements in the other allele. A putative second mutation could be located farther than 1427 basepairs upstream of the initiation codon, thus potentially affecting the normal expression of this gene or within intronic regions, generating an alternative aberrant splicing site. These are possibilities that remain to be elucidated. The present findings, which describe the novel missense mutation Y254D in the human type II 3 beta HSD gene, provide useful information on the structure-activity relationships of the 3 beta HSD superfamily.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Molecular basis of congenital adrenal hyperplasia in two siblings with classical nonsalt-losing 3 beta-hydroxysteroid dehydrogenase deficiency.

We report mutations of the type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene in two siblings, male and female, with congenital adrenal hyperplasia caused by classical nonsalt-losing 3 beta HSD deficiency. During childhood, the male sibling, born with ambiguous genitalia, and the female sibling, born with normal genitalia, both manifested symptoms of mild androgen excess; both apparently had normal zona glomerulosa function. Gonadal dynamic study at puberty showed the presence of partial gonadal 3 beta HSD deficiency in both siblings despite their spontaneous pubertal maturation. The 5'-region as well as exons I-II, III, and IV and portions of the adjacent introns of the type II 3 beta HSD gene were amplified by polymerase chain reaction and sequenced. In both siblings and their mother, an identical single nucleotide substitution mutation in intron III, six bases up-stream from exon IV, was identified in one allele. This mutation, G to A at nucleotide 6651, may create a new splicing junction and affect the normal splicing of the messenger ribonucleic acid. In the other allele of both siblings, a missense mutation from GGG (Gly) to AGG (Arg) at codon 129 (G129R) in exon IV was found. We assessed the effect of the G129R missense mutation on enzymatic activity by in vitro analysis of the mutant recombinant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 cells. Using homogenates from transfected cells, the G129R 3 beta HSD enzyme showed a Km value for pregnenolone of 10 +/- 2 mumol/L compared with 1.00 +/- 0.03 mumol/L for the wild-type type II 3 beta HSD enzyme. When dehydroepiandrosterone was used as substrate, the Km value for G129R3 beta HSD was 14 +/- 2 mumol/L compared with 2.1 +/- 0.2 mumol/L for the wild-type II 3 beta HSD enzyme. In addition to an apparent decrease in affinity, the G129R mutation caused a marked decrease in the apparent relative specific activity, thus leading to apparent relative specific efficiencies (relative specific activity/Km) of 2.0% and 4.7% that of the normal type II 3 beta HSD using pregnenolone or dehydroepiandrosterone as substrate, respectively. It appears likely that this low level of activity is sufficient to prevent salt loss, but it is also possible that part of the enzymatic activity comes from the putative remaining percentage of correctly spliced n6651 allele in these patients.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Guided tissue regeneration: comparison of bioabsorbable and non-bioabsorbable membranes. Histologic and histometric study in dogs.

This study examined histologic and histometric responses to 2 bioabsorbable membranes made from a synthetic copolymer of glycolide and lactide. They were tested for their biocompatibility, resorption characteristics, and ability to support periodontal regeneration. Expanded polytetrafluoroethylene (ePTFE) was used as control. Nine fox hound dogs with no periodontal disease were used. They were sedated and their teeth thoroughly scaled and root planed. Plaque control was maintained. Two weeks later, each dog was anesthetized using gas anesthesia. Buccal and lingual mucoperiosteal flaps were reflected in the mandibular premolar areas. Randomly selected, buccal alveolar bone was reduced on the 2nd and 4th premolars to a level 5 to 8 mm apical to the cemento-enamel junction creating a Class II buccal furcation defect on one quadrant, while the other quadrant received a Class II buccal defect only on the 4th premolar. Root surfaces were denuded of periodontal ligament and cementum, and notches were placed at the bone level of each root. In one quadrant, one site received Type I membrane and the other site received Type II. The contralateral quadrant received ePTFE. Flaps were positioned slightly coronally and sutured. Sutures were removed 1 week later. One month after surgery, 3 dogs were sacrificed and ePTFE barriers were removed from the remaining 6 dogs. Of these, 3 were sacrificed 3 months after surgery and the other 3 at 6 months. Undemineralized experimental tissues were embedded in methylmethacrylate and 8 to 10 microns thick sections were cut in a bucco-lingual direction throughout the mesiodistal extension of the tooth.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1994

Chordomas of the skull base. Radiologic and clinical evaluation.

Chordomas are uncommon skull base tumors, which are locally agressive and are usually not amenable to complete surgical resection. Proton beam irradiation, following surgery, is the preferred treatment modality. For diagnosis and determination of tumor site and extension, CT and MR imaging are the imaging modalities of choice. CT delineates bone destruction and the presence of calcifications and destroyed bone optimally. MR imaging is the modality of choice for better definition of the tumor margin from brain and other soft tissue structures (pharynx) and visualization of blood vessels. The signal intensities and enhancement pattern fail to differentiate chordoma from chondroid chordoma or chondrosarcoma. Chordomas arise from the clivus and therefore are located more centrally, whereas the majority of chondrosarcomas originate in the petroclival fissure and occur more laterally, although occasional overlap occurs in about one third of cases. Immunohistochemical methods allow differentiation of pure chordoma from chondroid chordoma and chondrosarcoma. Chordomas have a lower local control rate than chondrosarcomas.

Adult↗
PubMed1994

Left ventricular perforation by a pleural drainage tube. Report of a case with survival.

We present a case of survival as a result of surgical intervention after perforation of the left ventricle during the insertion of a pleural drainage tube. Surgical intervention was facilitated by the fact that the drainage tube responsible for the suspected perforation of the cardiac cavity was clamped and fastened, as opposed to withdrawn. The location of the perforation was established by measuring the pressure curve through the drainage tube.

Drainage↗
PubMed1994

A model for estimation of the pulmonary capillary pressure.

To estimate the pulmonary capillary pressure, a theory was introduced by Holloway and coworkers. Based upon this, a mathematical model describing the measured data was developed. Because the physiologic data are embedded in noise and the pulmonary capillary pressure cannot be measured directly, we simulated an extensive series of data. The noise properties of the data were as analyzed to design a signal-processing tool, that cancels the noise from the measured data. The signal processing tool developed for the current application consists of pre-processing with a moving time average filter and post-processing with a neural network. After a verification procedure the tool can be applied to measured data, hence a more reliable measurement of the pulmonary capillary pressure is achieved.

Fourier Analysis↗
PubMed1993

Structure and tissue-specific expression of a novel member of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family. The exclusive 3 beta-HSD gene expression in the skin.

Structures of cDNA clones encoding three members of the rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) family were characterized. To search for potential new types of 3 beta-HSD, rat types I and II 3 beta-HSD cDNAs were used as probes to screen a rat genomic DNA library. Among the clones isolated, one encodes a novel predicted rat 3 beta-HSD isoenzyme, chronologically designated type IV. The corresponding full-length cDNA was thereafter isolated by selective polymerase chain reaction amplification from rat ovary and day-15 placenta cDNA libraries. The rat type IV 3 beta-HSD cDNA encodes a predicted 372-amino acid protein of 41,854 daltons, which shares 90.9, 87.9, and 78.8% sequence identity with rat types I, II, and III proteins, respectively. Ribonuclease protection assay reveals that type IV 3 beta-HSD is the sole 3 beta-HSD mRNA species detectable in the skin and represents the predominant species in the placenta while being also detectable in the ovary and, to a lower degree, in the adrenal gland. Transient expression of type IV cDNA in SW-13 cells indicates 3 beta-HSD activity similar to that of rat type I 3 beta-HSD. The presence of multiple 3 beta-HSD genes should permit differential and tissue-specific regulation of this rate-limiting enzymatic activity essential in the biosynthesis of all classes of steroid hormones in both classical steroidogenic and intracrine peripheral tissues.

Amino Acid Sequence↗
PubMed1993

A rapid colorimetric in situ messenger RNA hybridization technique for analysis of epidermal growth factor receptor in paraffin-embedded surgical specimens of human colon carcinomas.

We have developed a rapid colorimetric in situ mRNA hybridization procedure to analyze epidermal growth factor receptor (EGF-R) transcripts in paraffin-embedded surgical specimens of human colon carcinomas. This technique is based on the use of 24-base oligonucleotide probes labeled with 6 biotin molecules at the 3' end. mRNA integrity was verified using a hyperbiotinylated 30-residue-long deoxythymidylate oligonucleotide probe, and the specificity of the reaction was confirmed by using labeled EGF-R-specific sense and antisense probes. Avidin alkaline phosphatase detection and the capillary technology used in the Microprobe System allowed for completion of the procedure in under 5 h. The human A431 epidermoid carcinoma cells growing in culture and fixed with formalin as well as paraffin-embedded sections of this tumor growing s.c. in nude mice served as positive controls. In situ hybridization with antisense EGF-R oligonucleotide probes directly correlated with EGF-R mRNA and protein levels observed by Northern blot and immunohistochemistry, respectively. In situ hybridization of paraffin-embedded sections of primary human colon carcinoma and metastases from liver and lymph node revealed cell-specific staining with EGF-R antisense oligonucleotide probes that correlated directly with Northern blot and immunohistochemistry analyses. Since this rapid and sensitive in situ mRNA hybridization technique can be used in properly preserved paraffin-embedded tissue, it allows for retrospective analyses of human tumor specimens using archival material.

Animals↗