Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb

Two unique forms of cytochrome P-450 (P-450), designated NMa and NMb, were recently isolated in this laboratory from nasal microsomes of rabbits. In the present study, polyclonal antibodies to the purified nasal cytochromes were prepared. Immunochemical analysis with specific rabbit anti-NMa and sheep anti-NMb antibodies indicated that P-450 isozymes identical to or having a high structural homology with NMa are present in both olfactory and respiratory mucosa, as well as in liver, but NMb was detected only in the olfactory mucosa. Neither form was detected in other tissues examined, including brain, esophageal mucosa, heart, intestinal mucosa, kidney, and lung. The specific occurrence of NMb in the olfactory mucosa was further substantiated by the detection and specific inhibition by anti-NMb of the formation of unique NMb-dependent metabolites of testosterone in olfactory microsomes but not in microsomes from liver or respiratory mucosa. Similar experiments with antibodies to previously purified rabbit hepatic P-450 isozymes indicated that not all of the hepatic cytochromes are expressed in the nasal tissues. Thus, P-450 isozymes structurally homologous to hepatic forms 2, 3a, and 4, but not 3b and 6, were found in the olfactory mucosa. On the other hand, only form 2 was detected in the respiratory mucosa. Immunoquantitation experiments revealed that NMa and NMb are the major P-450 forms in olfactory microsomes, whereas NMa and P-450 form 2 (or its homolog) constitute the major portion of the respiratory nasal microsomal P-450. The level of NMa in the liver is relatively low, accounting for less than 3% of total microsomal P-450 in this tissue. In addition, evidence is provided that NMa is the major catalyst in the dealkylation of two nasal carcinogens, hexamethylphosphoramide and phenacetin, in both olfactory and respiratory nasal microsomes.     

Long-term culture and functional characterization of follicular cells from adult normal human thyroids.

We have obtained long-term cultures of differentiated proliferating follicular cells from normal adult human thyroid glands. In vitro growth of such human cells has been sustained by a modified F-12 medium, supplemented with bovine hypothalamus and pituitary extracts and no added thyrotropin. Cultures have been expanded, cloned, frozen, successfully retrieved, and characterized. Functional characterization of these cells shows constitutive thyroglobulin production and release and thyrotropin-dependent adenosine 3',5'-cyclic monophosphate production, the latter apparently not associated with significant increases in DNA synthesis or cell proliferation. Genetic characterization of these cells by chromosome counting showed the normal diploid chromosome number. The ability to cultivate differentiated human thyroid follicular cells in long-term culture opens possibilities for investigating the transduction pathways of thyrotropin stimulation in normal and pathological human tissues, developing clinically relevant in vitro assays, and considering cellular and molecular therapies.     

Splenectomy for thrombocytopenia due to secondary hypersplenism

The response to splenectomy of patients with thrombocytopenia due to secondary hypersplenism is frequently unpredictable. Our experience indicated that splenectomy is seldom justified for this indication in patients with chronic myelogenous or chronic granulocytic leukemia. Since patients with chronic lymphocytic leukemia, hairy-cell leukemia, and stage IV lymphoma may have a more prolonged life expectancy, removal of the spleen brings about a satisfactory response of thrombocytopenia in some instances. Elevation of platelet counts after splenectomy in patients with agnogenic myeloid metaplasia is most likely to occur in women with the primary form of the disease. In other nonmalignant conditions, splenectomy has resulted in a satisfactory response in the majority of patients.     

44Sacral extracorporeal magnetic stimulation is an effective treatment for pelvic floor dyssynergia; Comparative study with biofeedback therapy

Background: Recent development of extracorporeal magnetic stimulation (ECMS) which uses current‐changing magnetic fields allows the induction of electrical stimulation in the desired deep tissue. Recent study showed the sacral nerve stimulation reduces corticoanal excitability that may play a functional role in anal continence mechanisms. Preliminary study shows that ECMS of sacral nerve can modify pelvic floor function and expel rectal balloon in patients with pelvic floor dyssynergia (PFD). Aims: To evaluate the effect of ECMS compared with biofeedback therapy (BF) in patients with PFD. Methods and Materials: Thirty‐eight patients who fulfilled Rome II criteria for PFD by colon transit time and anorectal function tests, were randomly treated with 8 sessions of ECMS (2/weeks; n = 19) at prone position or BF (2/weeks; n = 19) at sitting position. Stimulation parameters were set at 50–80% of maximum intensity, 10 and 50 Hz frequency, 3 s burst length with 3 and 6 s off using arm‐typed stimulator (BioCom‐1000, Mcube Co., Korea). Symptom scores for constipation with/without anorectal function test were repeatedly measured after each treatment. Response was defined as 50% or more decreased symptom score after treatment (partial response: 30–50%, poor: <30%). Results: Fifteen patients (age 49.1 ± 13.4 years, mean ± SD; 4 men) completed 8 session of BF and 14 patients (54.5 ± 17.6 years, 3 men) completed 8 session of ECMS. Four patients of BF group discontinued treatment due to unsatisfactory therapeutic effect (n = 1) and withdrew consent (n = 3) and 5 patients of ECMS group discontinued treatment because of same reasons (n = 1, 4). Total symptom scores were significantly decreased after treatment of 8 session in both treatment groups (13.4 ± 6.6 vs. 4.3 ± 4.0 for BF, p = 0.009; 14.9 ± 5.6 vs. 3.4 ± 4.0 for ECMS, p < 0.001). Bowel movements per week were also significantly increased after treatment in both groups (median 2 vs. 7 for BF, p = 0.035; median 2 vs. 7 for ECMS, p = 0.008). Thirteen out of 15 patients showed response in BF group and 12 out of 14 showed good response in ECMS group. No adverse effects in both groups. Conclusions: ECMS is as effective as BF for the treatment of PFD. Long‐term effect of ECMS for the patients with pelvic floor dyssynergia need to be evaluated in the near future.     

Bovine parathyroid cells: cultures maintained for more than 140 population doublings.

Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release.     

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.     

Increased frequency of congenital chromosomal aberrations in female partners of couples undergoing intracytoplasmic sperm injection

We evaluated the frequency of congenital chromosomal aberrations in a sample of 305 couples included in an intracytoplasmic sperm injection (ICSI) programme. Twenty individuals (3.3%) with congenital chromosomal abnormalities could be identified. The following types of abnormalities were observed: reciprocal translocations (n = 7), Robertsonian translocations (n = 3), inversions (n = 3), other structural aberrations (n = 4) and sex chromosome aberrations (n = 3). The rate of chromosomally abnormal males (10/305, 3.3%) lay within the expected range for patients with reduced semen quality. Surprisingly, 50% (10/20) of all abnormal karyotypes were contributed by the female partner of ICSI patients. These data confirm the higher incidence of chromosomal aberrations in infertile populations as compared with the baseline population risk. Additionally, the data imply that in some cases of male factor infertility a hidden female chromosomal factor may be present, which cannot be identified by standard clinical evaluation. In conclusion, we recommend chromosomal analysis in both partners of couples undergoing ICSI treatment.      

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.