The effect of peri-operative dexmedetomidine on the incidence of postoperative delirium in cardiac and non-cardiac surgical patients: a randomised, double-blind placebo-controlled trial.

Delirium occurs commonly following major non-cardiac and cardiac surgery and is associated with: postoperative mortality; postoperative neurocognitive dysfunction; increased length of hospital stay; and major postoperative complications and morbidity. The aim of this study was to investigate the effect of peri-operative administration of dexmedetomidine on the incidence of postoperative delirium in non-cardiac and cardiac surgical patients. In this randomised, double-blind placebo-controlled trial we included 63 patients aged ≥ 60 years undergoing major open abdominal surgery or coronary artery bypass graft surgery with cardiopulmonary bypass. The primary outcome was the incidence of postoperative delirium, as screened for with the Confusion Assessment Method. Delirium assessment was performed twice daily until postoperative day 5, at the time of discharge from hospital or until postoperative day 14. We found that dexmedetomidine was associated with a reduced incidence of postoperative delirium within the first 5 postoperative days, 43.8% vs. 17.9%, p = 0.038. Severity of delirium, screened with the Intensive Care Delirium Screening Checklist, was comparable in both groups, with a mean maximum score of 1.54 vs. 1.68, p = 0.767. No patients in the dexmedetomidine group died while five (15.6%) patients in the placebo group died, p = 0.029. For patients aged ≥ 60 years undergoing major cardiac or non-cardiac surgery, we conclude that the peri-operative administration of dexmedetomidine is associated with a lower incidence of postoperative delirium.

Role of choline PET/CT in guiding target volume delineation for irradiation of prostate cancer.

Choline PET/CT has shown limitations for the detection of primary prostate cancer and nodal metastatic disease, mainly due to limited sensitivity and specificity. Conversely in the restaging of prostate cancer recurrence, choline PET/CT is a promising imaging modality for the detection of local regional and nodal recurrence with an impact on therapy management. This review highlights current literature on choline PET/CT for radiation treatment planning in primary and recurrent prostate cancer. Due to limited sensitivity and specificity in differentiating between benign and malignant prostatic tissues in primary prostate cancer, there is little enthusiasm for target volume delineation based on choline PET/CT. Irradiation planning for the treatment of single lymph node metastases on the basis of choline PET/CT is controversial due to its limited lesion-based sensitivity in primary nodal staging. In high-risk prostate cancer, choline PET/CT might diagnose lymph node metastases, which potentially can be included in the conventional irradiation field. Prior to radiation treatment of recurrent prostate cancer, choline PET/CT may prove useful for patient stratification by excluding distant disease which would require systemic therapy. In patients with local recurrence, choline PET/CT can be used to delineate local sites of recurrence within the prostatic resection bed allowing a boost to PET-positive sites. In patients with lymph node metastases outside the prostatic fossa and regional metastatic lymph nodes, choline PET/CT might influence radiation treatment planning by enabling extension of the target volume to lymphatic drainage sites with or without a boost to PET-positive lymph nodes. Further clinical randomized trials are required to assess treatment outcomes following choline-based biological radiation treatment planning in comparison with conventional radiation treatment planning.

[18F]-florbetaben PET/CT Imaging in the Alzheimer's Disease Mouse Model APPswe/PS1dE9.

BACKGROUND: Positron-emission-tomography (PET) using 18F labeled florbetaben allows noninvasive in vivo-assessment of amyloid-beta (Aß), a pathological hallmark of Alzheimer's disease (AD). In preclinical research, [18F]-florbetaben-PET has already been used to test the amyloid-lowering potential of new drugs, both in humans and in transgenic models of cerebral amyloidosis. The aim of this study was to characterize the spatial pattern of cerebral uptake of [18F]-florbetaben in the APPswe/ PS1dE9 mouse model of AD in comparison to histologically determined number and size of cerebral Aß plaques. METHODS: Both, APPswe/PS1dE9 and wild type mice at an age of 12 months were investigated by smallanimal PET/CT after intravenous injection of [18F]-florbetaben. High-resolution magnetic resonance imaging data were used for quantification of the PET data by volume of interest analysis. The standardized uptake values (SUVs) of [18F]-florbetaben in vivo as well as post mortem cerebral Aß plaque load in cortex, hippocampus and cerebellum were analyzed. RESULTS: Visual inspection and SUVs revealed an increased cerebral uptake of [18F]-florbetaben in APPswe/ PS1dE9 mice compared with wild type mice especially in the cortex, the hippocampus and the cerebellum. However, SUV ratios (SUVRs) relative to cerebellum revealed only significant differences in the hippocampus between the APPswe/PS1dE9 and wild type mice but not in cortex; this differential effect may reflect the lower plaque area in the cortex than in the hippocampus as found in the histological analysis. CONCLUSION: The findings suggest that histopathological characteristics of Aß plaque size and spatial distribution can be depicted in vivo using [18F]-florbetaben in the APPswe/PS1dE9 mouse model.

Qualitative and quantitative differences of cytoskeleton proteins in embryos produced in vitro, in vivo, and by somatic nuclear transfer.

The cytoskeleton, consisting of complex and dynamic systems of structural filaments, intermediate filaments and microtubules, is not only a structural element but also contributes to many cellular processes such as functional compartments, transportation, mitosis, secretion, formation of cell extensions, and intercellular communication. Suggestions in rat 2-cell embryos that abnormal distributions of cytoskeletal proteins occurred following the initiations of developmental arrest and our former studies showing reduced intercellular contact zones in cloned bovine embryos prompted us to conduct comparative studies on 8-cell stage bovine embryos from nuclear transfer (NT), in vitro, and in vivo production. Immunohistochemistry and Laser-Scanning-Microscopy facilitated detection of cytoskeleton proteins--alpha-tubulin, F-actin, beta-catenin, and the cell adhesion protein cadherin; image and cluster analysis were subsequently used to study the distribution pattern of the proteins, whereas Western blot was carried out for their qualitative and quantitative analysis. The maximum fluorescence intensity of stained alpha-tubulin was observed in the cloned and the in vitro embryos. A significant higher intensity of staining for F-actin was observed in the in vivo and in vitro embryos. In contrast, Western blot revealed no differences of actin, tubulin, and catenin between the three tested groups whereas a lower abundance of cadherin proteins in the cloned embryos was visible. The distribution of actin filaments in cloned embryos was more centric or one-sided and not peripheral whereas the stained spots of catenin were smaller in comparison to in vivo or in vitro produced embryos. These differences recorded in the distribution patterns may be associated with cell physiological processes related to an influenced actin-catenin-cadherin system. In conclusion, reduced intercellular contacts coupled with abnormal distribution of cytoskeletal proteins seem to play an important role in the developmental arrest encountered normally at the 8-cell stage in bovine cloned embryos.

External radiation exposure, excretion, and effective half-life in 177Lu-PSMA-targeted therapies.

BACKGROUND: Prostate-specific membrane antigen (PSMA)-targeted therapy with 177Lu-PSMA-617 is a therapeutic option for patients with metastatic castration-resistant prostate cancer (mCRPC). To optimize the therapy procedure, it is necessary to determine relevant parameters to define radiation protection and safety necessities. Therefore, this study aimed at estimating the ambient radiation exposure received by the patient. Moreover, the excreted activity was quantified. RESULTS: In total, 50 patients with mCRPC and treated with 177Lu-PSMA-617 (mean administered activity 6.3 ± 0.5 GBq) were retrospectively included in a bi-centric study. Whole-body dose rates were measured at a distance of 2 m at various time points after application of 177Lu-PSMA-617, and effective half-lives for different time points were calculated and compared. Radiation exposure to the public was approximated using the dose integral. For the estimation of the excreted activity, whole body measurements of 25 patients were performed at 7 time points. Unbound 177Lu-PSMA-617 was rapidly cleared from the body. After 4 h, approximately 50% and, after 12 h, approximately 70% of the administered activity were excreted, primarily via urine. The mean dose rates were the following: 3.6 ± 0.7 µSv/h at 2 h p. i., 1.6 ± 0.6 µSv/h at 24 h, 1.1 ± 0.5 µSv/h at 48 h, and 0.7 ± 0.4 µSv/h at 72 h. The mean effective half-life of the cohort was 40.5 ± 9.6 h (min 21.7 h; max 85.7 h). The maximum dose to individual members of the public per treatment cycle was ~ 250 ± 55 µSv when the patient was discharged from the clinic after 48 h and ~ 190 ± 36 µSv when the patient was discharged after 72 h. CONCLUSIONS: In terms of the radiation exposure to the public, 177Lu-PSMA is a safe option of radionuclide therapy. As usually four (sometimes more) cycles of the therapy are performed, it must be conducted in a way that ensures that applicable legal requirements can be followed. In other words, the radiation exposure to the public and the concentration of activity in wastewater must be sub-marginal. Therefore, in certain countries, hospitalization of these patients is mandatory.

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley.

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.

Interaction Analyses of Genes Required for Resistance Responses to Powdery Mildew in Barley Reveal Distinct Pathways Leading to Leaf Cell Death.

Race-specific resistance in barley to the powdery mildew fungus (Erysiphe graminis f sp hordei) is associated with a cell death reaction (hypersensitive response [HR]). Genetically, it is dependent on dominant resistance genes (Mlx), and in most cases, it is also dependent on Rar1 and Rar2. Non-race-specific resistance to the fungus, which is due to the lack of the Mlo wild-type allele, is dependent on Ror1 and Ror2 and is not associated with an HR in the region of pathogen attack. However, the absence of the Mlo wild-type allele stimulates a spontaneous cell death response in foliar tissue. This response is also controlled by Ror1 and Ror2, as indicated by trypan blue staining patterns. Lack of Mlo enhances transcript accumulation of pathogenesis-related genes upon fungal challenge, and this response is diminished by mutations in Ror genes. Using DNA marker-assisted selection of genotypes, we provide evidence, via gene interaction studies, that Ror1 and Ror2 are not essential components of race-specific resistance and do not compromise hypersensitive cell death. Reciprocal experiments show that neither is Rar1 a component of mlo-controlled resistance nor does it affect spontaneous cell death. We show that mlo- and Ror-dependent resistance is active when challenged with E. g. f sp tritici, a nonhost pathogen of barley. Our observations suggest separate genetic pathways operating in race-specific and non-race-specific resistance; they indicate also a separate genetic control of hypersensitive and spontaneous cell death in foliar tissue.

Measurement of redox potential and steroid concentrations in the follicular fluid of growing and regressing follicles of mares.

The aim of this study was to prove if oxidation-reduction levels in the follicular fluid were new functional indices of follicular health and whether there was a high level of accordance with endocrinological parameters and with the growth stage as detected by ultrasound monitoring of individual follicles during the oestrous cycle in mares. Follicles were classified as growing and regressing follicles using ultrasonography. Altogether 48 follicles with a diameter from 20 to 56 mm were aspirated by transvaginal ultrasound guided follicular aspiration. Follicular concentration of oestradiol and progesterone in relation to the diameter of growing follicles showed correlations of r = 0.64 and r = 0.57, respectively. The redox potential derived index D2 varied from -448 to +431 in the collected fluids of the follicles. The accordance of the judgement of all follicles using both complexes of methods - endocrinological and ultrasonographic parameters vs. analysis of oxidation and reduction levels - reached 72.5%. This finding has shown that parameters of redox reactions do not correlate closely with the stage of follicular growth or regression as determined by in vivo scanning of ovaries or by assessment of follicular steroid concentrations. However, the measurement of redox potentials offers an opportunity to examine the whole process of metabolism in follicular cells and to forecast impairments of cellular performances. Changes of redox parameters in growing follicles enable an earlier prediction of their further development. The data demonstrate that growing and regressing follicles do not represent nonatretic, early atretic and atretic follicles, respectively.

The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley.

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.

The impact of pharmacogenetics on the future of healthcare.

Pharmacogenetics has been promoted as potentially providing benefits to patients, managed care organizations and pharmaceutical companies. This has not translated into products that benefit healthcare developers, providers or consumers. The reasons for this are many, but this will change as the financial incentives become clear for the pharmaceutical industry to develop products that use genetic susceptibility as part of the rationale for products, healthcare providers have increasing incentive to reduce costs, and patients demand up-to-date technologies to optimize healthcare. Recent studies have established genetic contributions that alter the response to therapy for some disease entities, and more will follow as pharmacogenetics becomes increasingly accepted as an important consideration in the therapeutic decision-making process.

Variant cytochrome P450 CYP2D6 allelic frequencies in Parkinson's disease.

Aberrant detoxification of environmental agents may be the basis for an inherited predisposition to Parkinson's disease. A CYP2D6 genetic marker of the debrisoquine hydroxylase "poor metabolizer" phenotype was found to be significantly increased in Parkinson's disease patients compared to controls, as has been shown in previous studies. Presence of this marker gives an odds ratio of 1.86 for Parkinson's disease (95% confidence interval 1.33-2.39, P < 0.02). For comparison, a CYP1A1 polymorphism, which is not known to be associated with aberrant drug metabolism, showed no association with Parkinson's disease in our study.

Angiotensin II type-2 (AT2) receptor-mediated inhibition of NMDA receptor signalling in neuronal cells.

The N-methyl-D-aspartate (NMDA) receptor has been reported to be important in synaptic plasticity, neuronal development, normal brain function and neurologic disease. We have recently shown that PC12W cells, a subclone of rat pheochromocytoma PC12 cell line, release nitric oxide (NO), as measured by in vitro spin-trapping combined with electron paramagnetic resonance (EPR) spectroscopy, when challenged with NMDA [Norby, S.W., Weyhenmeyer, J.A. and Clarkson, R.B., Stimulation and inhibition of NO production in macrophages and neuronal cells as observed by spin trapping, Free Rad. Biol. Med., 22 (1997) 1-9]. In the present study, we provide immunochemical evidence for the expression of both the NMDAR1 and NMDAR2A/B receptor subunits in PC12W cells, that express only the angiotensin type-2 (AT2) receptor subtype, and in NG108-15 (NG108) cells, a murine neuroblastoma x glioma hybrid that expresses both the angiotensin type-1 (AT1) and AT2 receptor subtypes. We also show that treatment of PC12W cells with angiotensin (Ang II) decreases NMDA-induced NO release by 28.0 +/- 4.2%, and that this response can be attenuated by pre-treating the cells with the isoform-specific AT2 antagonist, PD 123319. Interestingly, there was no effect on cGMP accumulation in PC12W cells treated with NMDA. Similar experiments were carried out using NG108 cells since the binding properties and functional characteristics of their NMDA receptors have been previously described [Ohkuma, S., Katsura, M., Chen, D., Chen, S. and Kuriyama, K., Presence of N-methyl-D-aspartate (NMDA) receptors in neuroblastoma x glioma hybrid NG 108-15 cells-analysis using 45Ca2+ influx and [3H]MK-801 binding as functional measures, Mol. Brain Res. 22 (1994) 166-172]. Our results show that NG108 cells significantly increase cGMP levels when challenged with NMDA (21.2 +/- 5.0% over control levels), and that this response can be attenuated by the addition of angiotensin (57.1 +/- 6.2% of stimulated levels). The effect of angiotensin on NMDA-mediated changes in cGMP levels was blocked by the AT2 antagonist, PD 123319, but was not significantly changed by the addition of the AT1 antagonist, losartan. Further, Ang II action on NMDA signalling in NG108 cells was completely inhibited by the addition of both the AT1 and AT2 antagonists. Taken together, these results suggest that AngII inhibits NMDA-mediated NO and cGMP production through a mechanism involving the AT2 receptor subtype.

Identification of a new polymorphism in the human proteolipid protein gene.

A polymorphism in the gene for proteolipid protein has been identified, using amplification by the polymerase chain reaction, restriction enzyme digestion, and fragment separation by polyacrylamide gel electrophoresis. The polymorphism is located in the transcribed 3'-untranslated region, a region with potential regulatory signals. The mutation consists of a single base pair insertion into a Hae III restriction site, producing a larger rare fragment of 409 bp as compared with the more frequent 325 bp fragment. The gene for proteolipid protein is on the X chromosome; thus the males are hemizygous for the rare allele and the females are heterozygous carriers. The polymorphism occurs with a frequency of 0.046 in a population of European origin and also has a rare frequency in multiple sclerosis patients.

Genetic predisposition plays a role in nigral cell death in Parkinson's disease.

Both genetic and environmental factors are involved in the etiology of Parkinson's disease (PD). The recent identification of specific autosomal genes that lead to variants of PD confirms that genetic factors are important. Identifying and confirming other genetic factors responsible for PD is a difficult task because PD is a complex disease, the results of multiple genetic and environmental factors leading to a final common pathology. This review will discuss how advances in human genetics will allow future unraveling of the complex interactions between genetics and environment in the etiology of PD.

Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments.

Endoscopically and ultrasonographically guided ovum pick up were studied in 15 heifers once per week. Althogether 60 sessions were carried out. The number of aspirated follicles per animal (13.0 vs 10.3) did not differ significantly between both methods. Similar recovery rates (39 % vs 44 %) were achieved after repeated sessions. As a consequence the number of recovered oocytes per animal (5.2 vs 4.7) were not significantly different. Inter-animal- and intra-animal-variations were more important for the results than the applied methods. In contrast to these findings the quality of recovered cumulus-oocyte-complexes (COC) was significantly influenced by the methods. The COC were divided into 4 categories a) oocytes with compact cumulus, b) oocytes with rest of compacted cumulus, c) oocytes with expanded cumulus and d) oocytes without cumulus. There was a higher denudation rate of COC when using the endoscopic aspiration (62.0 % vs 6.6 %) because of turbulent current in this aspiration system. Advantages and disadvantages of both methods are described and discussed. Generally, the ultra sonographic method is the less traumatic procedure for the vagina, fornix and for abdominal organs. The other method is less traumatic for the ovaries.

Assessing prostate cancer growth with citrate measured by intact tissue proton magnetic resonance spectroscopy.

BACKGROUND: Annual PSA tests have led to a significant increase in the number of prostate cancer (PCa) cases diagnosed. This increased incidence has led to overtreatment of many patients, as current pathology often cannot distinguish latent from aggressive PCa. Studies have shown that the depletion of zinc in prostate cells correlated with cell-line growth rates, and may therefore relate to the progression of PCa. Furthermore, as zinc is normally an inhibitor of citrate oxidation, the reduction of zinc in PCa may cause a decrease in citrate secretion levels in the glandular epithelia of PCa patients. METHODS: Using high-resolution magic angle spinning proton magnetic resonance spectroscopy followed by quantitative histopathology, we investigate unit histo-benign prostate epithelial citrate concentrations in intact tissue samples obtained from 18 patients with pre-surgical PSA values less than 20 ng/ml. Using these data, we evaluate correlations between citrate concentrations and PSA velocities, densities and blood percent-free PSA. RESULTS: We observe different linear patterns between citrate concentrations and histo-benign glandular epithelia from patients of different PSA velocities. More importantly, we obtain a significant correlation between PSA velocity, density and percent-free PSA, and citrate concentrations in unit volume of histo-benign epithelial glands of the peripheral zone. CONCLUSIONS: Low levels of citrate in unit volume represent rapidly increasing PSA values, and, therefore, may be used as an indicator of fast-growing PCa. Thus, tissue samples obtained at the time of biopsy may be evaluated for their citrate concentrations for the prediction of PCa growth rates, allowing for the implementation of alternative treatment options and reducing overtreatment.

[Method for the photometric determination of aldehyde oxidase activity]. / Methode zur photometrischen Bestimmung der Aktivität von Aldehydoxydase.

A method has been developed for the determination of aldehyde oxidase. Dimethylaminocinnamaldehyde was taken as substrate. It is oxidized to the corresponding acid and changes ist absorption at 398 nm. Except dissolved oxygen no additional electron acceptor is needed. This alternative assay was found to be about twice as sensitive compared to the method with acetaldehyde and dichlorophenol indophenol. Aldehyde oxidase activity was estimated in the raw extract of livers of pig, sheep and rat. Xanthine oxidase does not interfere.

A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range.

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.

Steroid receptors in Streptomyces hydrogenans: isolation and characterization of a high affinity receptor for 5alpha-dihydrotestosterone.

There are different steroid binding receptors in the cytosol of Streptomyces hydrogenans. A high molecular weight component binds 5alpha-dihydrotestosterone (17beta-hydroxy-5alphaH-androstan-3-one) with high affinity. Partial purification is achieved by density gradient centrifugation and filtration on Sephadex G-200. 5alpha-Dihydrotestosterone and progesterone compete for the same binding sites on the receptor. Cyproterone and testosterone are not bound. As it is easy to isolate the binding fraction, a simple assay for 5alpha-dihydrotestosterone or progesterone in biological samples is available.

Human placental sphingomyelinase. Purification to homogeneity, antigenic properties and partial amino-acid sequences of the enzyme.

Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.