Rare compound heterozygous variants in PNKP identified by whole exome sequencing in a German patient with ataxia-oculomotor apraxia 4 and pilocytic astrocytoma.

Ataxia-oculomotor apraxia type 4 (AOA4) is a rare autosomal recessive neurologic disorder. The phenotype is characterized by ataxia, oculomotor apraxia, peripheral neuropathy and dystonia. AOA4 is caused by biallelic pathogenic variants in the PNKP gene encoding a polynucleotide kinase 3'-phosphatase with an important function in DNA-damage repair. By whole exome sequencing, we identified 2 variants within the PNKP gene in a 27-year-old German woman with a clinical AOA phenotype combined with a cerebellar pilocytic astrocytoma diagnosed at 23 years of age. One variant, a duplication in exon 14 resulting in the frameshift c.1253_1269dup p.(Thr424fs*49), has previously been described as pathogenic, for example, in cases of AOA4. The second variant, representing a nonsense mutation in exon 17, c.1545C>G p.(Tyr515*), has not yet been described and is predicted to cause a loss of the 7 C-terminal amino acids. This is the first description of AOA4 in a patient with central European descent. Furthermore, the occurrence of a pilocytic astrocytoma has not been described before in an AOA4 patient. Our data demonstrate compound heterozygous PNKP germline variants in a German patient with AOA4 and provide evidence for a possible link with tumor predisposition. Localization of the 2 variants in human PNKP NP_009185.2. NM_007254.3:c.1253_1269dup p.(Thr424fs*49) is predicted to cause a frameshift within the kinase domain, NM_007254.3:c.1545C>G p.(Tyr515*) is predicted to cause loss of 2 C-terminal amino acids of the kinase domain and 5 additional C-terminal amino acids.

Factors associated with thrombosis in pediatric patients with systemic lupus erythematosus.

OBJECTIVE: The risk of thrombosis is increased in patients with systemic lupus erythematosus (SLE). Few studies have assessed factors associated with thrombosis within the pediatric SLE (pSLE) population. We sought to better characterize these associated factors in pSLE patients using the Childhood Arthritis & Rheumatology Research Alliance (CARRA) registry. METHODS: Within the CARRA registry, patients with a history of thrombosis were compared to those without. Univariate logistic regression was used to calculate odds ratios. A multivariable logistic regression model was conducted that included variables from the univariate analysis that had a p value < 0.10 and other variables identified as clinically significant from published literature. RESULTS: Among the 979 pSLE patients in the CARRA registry, 24 (2.5%) patients had a history of arterial thrombosis and 35 (3.6%) of venous thrombosis. In the univariate analysis, the odds ratio of having a thrombotic event were found to be significantly higher in patients with a history of vasculitis, avascular necrosis (AVN), or antiphospholipid antibody (aPL). Similar results were found for vasculitis, AVN, and aPL in the multivariable analysis. CONCLUSION: Our study of pSLE patients suggests that vasculitis, positive APL, and AVN are associated with thrombotic events in this population.

Loss of function of PGAP1 as a cause of severe encephalopathy identified by Whole Exome Sequencing: Lessons of the bioinformatics pipeline.

We evaluated a multiple consanguineous Turkish family with two children, a boy and a girl, affected by severe encephalopathy, hypotonia, microcephaly and retinal dystrophy by a combination of linkage analysis and Whole Exome Sequencing (WES). We analyzed the sequence data by two different bioinformatics pipelines which did not differ in overall processing strategy but involved differences in software used, minor allele frequency (MAF) thresholds and reference data sets, the usage of in-house control exomes and filter settings to prioritize called variants. Assuming autosomal recessive mode of inheritance, only homozygous variants present in both children were considered. The resulting variant lists differed partially (nine variants identified by both pipelines, ten variants by only one pipeline). Major reasons for this discrepancy were different filters for MAF and different variant prioritizations. Combining the variant lists with the results of linkage analysis and further prioritization by expression data and prediction tools, an intronic homozygous splice variant (c.1090-2A>G; IVS9-2A>G; p.?) in PGAP1 (Post-GPI Attachment To Proteins 1) was identified and validated by cDNA analysis. PGAP1 ensures the first step of maturation of GPI (glycosylphosphatidylinositol)-anchor proteins. Recently, a homozygous loss-of-function mutation in PGAP1 has been reported in one family with two children affected by a similar phenotype. The present report not only illustrates the possible influence of specific filtering settings on the results of WES but also confirms PGAP1 as a cause of severe encephalopathy.

The accuracy of a patient or parent-administered bleeding assessment tool administered in a paediatric haematology clinic.

Classifying and describing bleeding symptoms is essential in the diagnosis and management of patients with mild bleeding disorders (MBDs). There has been increased interest in the use of bleeding assessment tools (BATs) to more objectively quantify the presence and severity of bleeding symptoms. To date, the administration of BATs has been performed almost exclusively by clinicians; the accuracy of a parent-proxy BAT has not been studied. Our objective was to determine the accuracy of a parent-administered BAT by measuring the level of agreement between parent and clinician responses to the Condensed MCMDM-1VWD Bleeding Questionnaire. Our cross-sectional study included children 0-21 years presenting to a haematology clinic for initial evaluation of a suspected MBD or follow-up evaluation of a previously diagnosed MBD. The parent/caregiver completed a modified version of the BAT; the clinician separately completed the BAT through interview. The mean parent-report bleeding score (BS) was 6.09 (range: -2 to 25); the mean clinician report BS was 4.54 (range: -1 to 17). The mean percentage of agreement across all bleeding symptoms was 78% (mean κ = 0.40; Gwet's AC1 = 0.74). Eighty percent of the population had an abnormal BS (defined as ≥2) when rated by parents and 76% had an abnormal score when rated by clinicians (86% agreement, κ = 0.59, Gwet's AC1 = 0.79). While parents tended to over-report bleeding as compared to clinicians, overall, BSs were similar between groups. These results lend support for further study of a modified proxy-report BAT as a clinical and research tool.

Multiplexed Imaging Strategy to Distinguish Indeterminant Biliary Strictures: An Ex Vivo Study.

Introduction: Indeterminant biliary strictures can be either malignant or benign. Biliary intraepithelial neoplasia (BilIN) is the precursor lesion to cholangiocarcinoma, a deadly bile duct cancer. Current diagnostic methods are limited by inadequate amounts of cells and tissues collected. Aim: We aim to demonstrate use of fluorescently-labeled peptides specific for EGFR, claudin-1, and ErbB2 to perform multiplexed imaging of biliary neoplasia. Methods: Formalin fixed and paraffin embedded specimens resected from human biliary strictures were sectioned. A gastrointestinal pathologist used standard criteria to score immunohistochemistry from biliary neoplasia and adjacent normal epithelium from the same specimen. Peptides specific for EGFR, claudin-1, and ErbB2 were fluorescently-labeled with FITC, Cy5, and IRDye800, respectively. The fluorophores were chosen to provide spectral separation to distinguish the individual targets. Immuno fluorescence images were collected using confocal microscopy. Results: Target expression was validated using immunohistochemistry. Staining was visualized on the surface of biliary duct epithelial cells and not in the stroma. Greater fluorescence intensity was observed for peptide binding to biliary neoplasia by comparison with normal. The mean ratio for neoplasia-to-normal was 1.4, 1.7, and 1.6, respectively, and the average intensities were significantly greater for neoplasia than normal for each peptide. Peptides and antibody binding co-localized with correlation of ρ=0.64, 0.51 and 0.62, respectively. Conclusions: A panel of fluorescently-labeled peptides can distinguish BilIN and cholangiocarcinoma from normal biliary epithelium, and may be used for multiplexed imaging of indeterminant biliary strictures.

Mercury loading within the Selenga River basin and Lake Baikal, Siberia.

Mercury (Hg) loading in Lake Baikal, a UNESCO world heritage site, is growing and poses a serious health concern to the lake's ecosystem due to the ability of Hg to transform into a toxic form, known as methylmercury (MeHg). Monitoring of Hg into Lake Baikal is spatially and temporally sparse, highlighting the need for insights into historic Hg loading. This study reports measurements of Hg concentrations from water collected in August 2013 and 2014 from across Lake Baikal and its main inflow, the Selenga River basin (Russia, Mongolia). We also report historic Hg contamination using sediment cores taken from the south and north basins of Lake Baikal, and a shallow lake in the Selenga Delta. Field measurements from August 2013 and 2014 show high Hg concentrations in the Selenga Delta and river waters, in comparison to pelagic lake waters. Sediment cores from Lake Baikal show that Hg enrichment commenced first in the south basin in the late-19th century, and then in the north basin in the mid-20th century. Hg flux was also 20-fold greater in the south basin compared to the north basin sediments. Hg enrichment was greatest in the Selenga Delta shallow lake (Enrichment Ratio (ER) = 2.3 in 1994 CE), with enrichment occurring in the mid-to late-20th century. Local sources of Hg are predominantly from gold mining along the Selenga River, which have been expanding over the last few decades. More recently, another source is atmospheric deposition from industrial activity in Asia, due to rapid economic growth across the region since the 1980s. As Hg can bioaccumulate and biomagnify through trophic levels to Baikal's top consumer, the world's only truly freshwater seal (Pusa sibirica), it is vital that Hg input at Lake Baikal and within its catchment is monitored and controlled.

Yeast identification in grape juice concentrates from Argentina.

AIMS: The purpose of this study was to identify yeast species present in spoiled and unspoiled grape juice concentrates from Argentine industries. METHODS AND RESULTS: Osmophilic and osmotolerant yeasts were isolated from spoiled--visually effervescent--and unspoiled--without any visible damage--grape juice concentrates by the spread-plate technique in two culture media. Yeast identification was done by classical and molecular methods. Zygosaccharomyces rouxii was the only species isolated from spoiled grape juice concentrates. In unspoiled samples, five different species were identified: Z. rouxii was isolated at a higher frequency, followed in decreasing order by Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia anomala and Kluyveromyces delphensis. CONCLUSIONS: Yeasts isolated from grape juice concentrates were characterized by a limited taxonomic diversity, where Z. rouxii was the main species isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: Grape production in Argentina is mainly devoted to the industry where wine and grape juice concentrates represent major types of commercial products. Little information on common yeast contaminants is available for grape juice concentrates. This study constitutes the first report of osmophilic yeast species present in spoiled and unspoiled grape juice concentrates elaborated in Argentina.

Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation.

Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.

The haemodynamic effect of an adjustable band in an arteriovenous fistula.

A number of treatment approaches exist for excessive flowrate arteriovenous fistulae. Banding has a number of advantages, yet there is concern over its use due to reported high post-surgery thrombosis rates. A computational study is conducted of a new technique, to elucidate the hemodynamics present in the process. The key improvement involves the use of an adjustable band which can be used to optimise the flowrate during the surgery. The pressure and flowrate changes are apparent from the computational results and the computational results also demonstrate that further optimization may be possible. We then present a small cohort of five cases where the new banding procedure has been implemented with success. The new technique was combined with intra-operative ultrasound flow monitoring.

Cell populations and known surface antigens of tumors induced by Abelson virus in pristane-primed BALB/c mice: an analysis by immunoelectron microscopy;.

When Abelson murine leukemia virus (MuLV-A) was inoculated into pristane-primed BALB/c mice, either plasmacytomas (PCT), lymphosarcomas, or their mixed form was induced with a shorter latent period than that for PCT induction in mice treated with pristane alone. All 3 neoplasms were designated Abelson's tumors. The morphology of the induced malignant cells and the presence of known surface antigens were analyzed by electron microscopy. The cell surface antigens (CSA) examined were PC1, X.1, Gross (GCSA), and Moloney (MCSA); viral envelope antigens (VEA) were xVEA, x1-VEA, sub-gsVEA, and MVEA. MuLV-A-pristane-induced PCT cells produced markedly fewer intracisternal type-A particles than did mineral oil-induced PCT cells. Most, if not all, mineral oil-induced PCT cells carried PC1 antigen and produced many extracellular type-C viruses carrying xVEA and x1-VEA, whereas Abelson's tumor cells had both a lower incidence and a smaller amount of PC1 and X.1 antigens and xVEA+ virus and lacked x1-VEA+ virus. In addition, most Abelson's tumors and their type-C viruses did not carry MCSA and MVEA ro GCSA and sub-gsVEA. All the macrophages in the PC1+ Abelson's tumors and many macrophages in PC1-Abelson's tumors contained various amounts of PC1 on their cell surfaces. For these reasons, Abelson's tumors were clearly distinct from pristane- or mineral oil-induced BALB/c PCT and from M-MuLV- ro G-MuLV-induced tumors.

Location by immunoelectron microscopy of carcinoembryonic antigen on cultured adenocarcinoma cells.

The tissue-culture cell line HT-29, derived from a primary colon adenocarcinoma and previously shown to secrete carcinoembryonic antigen (CEA), was examined by immunoelectron microscopy for the location of CEA in relation to the cells. CEA was closely adjacent to the plasma membrane, a location indistinguishable from that of alloantigens and other tumor-associated antigens.

Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.

Effect of WEB 2086 on myocardial infarct size and regional blood flow in the dog.

OBJECTIVE: Systemic administration of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-phosphocholine) produces hypotension and decreased cardiac output; in isolated heart preparations PAF increases coronary vascular resistance and depresses inotropic state. A precursor of PAF bioactivity has been found early in myocardial ischaemia and other reports have suggested that PAF antagonists can reduce myocardial damage and ventricular arrhythmia. This study concerns the effects of WEB 2086, a PAF antagonist, on myocardial infarct size and coronary blood flow after total coronary artery occlusion. METHODS: Open chest anaesthetised dogs (n = 26) were pretreated with either WEB 2086 (5 mg.kg-1) or saline before proximal occlusion of the circumflex artery and constant infusion of WEB 2086 (1 mg.kg-1.h-1) or saline was maintained for 5 h. Cardiac output and regional myocardial flow were measured with radiolabelled microspheres (46Sc, 57Co, and 113Sn) before and immediately after occlusion and 5 h later. In the 22 dogs surviving occlusion, infarct size was determined by planimetry of cross sectional slices after exposure to triphenyltetrazolium chloride. RESULTS: Infarct size was not different between treated and control groups, at 23.6(SEM 2.3)% v 24.8(3.7)% of left ventricle, and was not different between groups when related to vasculature at risk and to collateral blood flow determined with microspheres. CONCLUSIONS: No beneficial effect of a relatively large dose of the potent PAF antagonist, WEB 2086, on myocardial infarct size or collateral blood flow was found after relatively short duration of myocardial ischaemia in the dog.

Whole blood aggregation, thromboxane release and the lyso derivative of platelet activating factor in myocardial infarction and unstable angina.

To investigate the pathophysiology of intracoronary thrombus formation we measured whole blood aggregation in response to ADP, platelet activating factor (PAF) and collagen, along with thromboxane B2 (TXB2) production during collagen induced aggregation, plasma TXB2 and plasma levels of lyso-PAF, in 38 subjects with and without ischaemic heart disease (12 with acute myocardial infarction, 9 with prolonged ischaemic chest pain without infarction and 17 normals). Lyso-PAF was measured, after in vitro acetylation to active PAF, by bioassay using 14C-serotonin labelled rabbit platelets. TXB2 was measured by radioimmunoassay. Plasma TXB2 was elevated at presentation only in patients with myocardial infarction (p less than 0.01). While impedance aggregation was similar in the three groups, aggregation to collagen resulted in greater release of TXB2 in subjects with myocardial infarction (p less than 0.01), an abnormality persisting 2-4 months later. Plasma lyso-PAF levels were significantly depressed throughout the first week in subjects with infarction (p less than 0.002), but after 2 to 4 months the level was greater than in normal subjects (p less than 0.001), changes presently unexplained. It is possible that the disorder of platelet function preceded and predisposed to coronary thrombosis. The findings strengthen the grounds for aspirin therapy in acute myocardial infarction.

Plasma platelet activating factor degradation and serum lipids after coronary bypass surgery.

OBJECTIVE: Platelet activating factor (PAF) is a potent mediator in inflammatory responses and maybe involved in various disease states. Degradation of PAF in plasma results from the action of a specific, lipoprotein associated, acetylhydrolase. The aim was to determine plasma acetylhydrolase activity under optimised conditions, PAF half life, phospholipase A2 activity, the lyso-derivative of PAF (lyso-PAF), and lipids in patients undergoing coronary artery bypass grafting. METHODS: The study variables were determined 3 d and 7 d following coronary artery surgery and compared to presurgical values in 15 males, age 55(SEM 4) years. RESULTS: Three days following coronary bypass grafting, total, LDL and HDL cholesterol fell significantly by 30%, 45%, and 15% respectively (p less than 0.001), all decreases correlating with bypass time (p less than 0.025). Concentrations remained low at 7 d (p less than 0.005). Acetylhydrolase activity fell by 38% (p less than 0.001) at 3 d post-surgery and remained depressed, but plasma PAF half life did not change after surgery. The inverse relationship between acetylhydrolase activity and plasma PAF half life preoperatively (p less than 0.01) was not evident after surgery. There was a direct linear relationship between acetylhydrolase activity and both total (p less than 0.002) and LDL cholesterol (p less than 0.001) before surgery. The fall in acetylhydrolase activity correlated with the fall in these lipids (p less than 0.01) but not with that of HDL cholesterol. Plasma lyso-PAF decreased by 65% (p less than 0.001) at 3 d and remained depressed (p less than 0.001). Plasma phospholipase A2 activity increased by 60% (p less than 0.01) and remained raised (p less than 0.05), the increase at 3 d being related to bypass time (p less than 0.05). CONCLUSIONS: The large fall in plasma acetylhydrolase activity after coronary bypass grafting is consistent with the fall in plasma lipids. However, the absence of a significant change in the measured PAF half life in plasma raises questions as to the pathophysiological significance of the decrease in acetylhydrolase activity.

Effect of chronic corticosterone application on depression-like behavior in C57BL/6N and C57BL/6J mice.

Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long-term increase in corticosterone levels. Thus, male mice from both sub-strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice.

Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality.

Insulin-like growth factor receptors and binding protein in rat neuroblastoma cells.

B104, an established rat neuroblastoma cell line exhibiting specific neuronal qualities, was chosen as a model to study insulin-like growth factor (IGF) binding and action in the central nervous system. Specific binding of [125I]IGF-II to B104 membranes averaged 12.2 +/- 4.0% (mean +/- SD)/100 micrograms/ml protein compared with [125I]IGF-I binding of 10.1 +/- 2.9%. In competitive binding studies employing [125I]IGF-II as the radioligand, high affinity for IGF-II was demonstrated (50% displacement at 2.7 ng/ml), with none for IGF-I or insulin. Upon cross-linking [125I]IGF-I to membranes under reducing conditions, two prominent bands were observed, migrating with apparent mol wt (Mr) of 135,000 and 280,000. Both bands were inhibited by IGFs and insulin, but not by R-II-PABI, a polyclonal antibody to the type 2 receptor. These bands presumably represent the alpha-subunit and an incompletely reduced alpha-alpha-dimer of the type 1 IGF receptor. When cross-linking [125I]IGF-II to membranes under reducing conditions, the primary labeled bands migrated with apparent Mr of 260,000 and 280,000. These bands were inhibited by IGF-II and R-II-PABI, but not by insulin, and probably represent the monomeric type 2 receptor. In addition, we observed a minor band at apparent Mr 35,000, which was inhibited by IGF but not by insulin. By a modified cross-linking technique, we confirmed the existence of a small IGF-binding protein in the serum-free conditioned medium of B104 cultures, migrating as two bands with apparent Mr of 33,000-39,000. These proteins demonstrated high affinity for IGF-I and IGF-II, but none for insulin. In summary, this study demonstrates the presence in B104 rat neuroblastoma cells of 1) abundant classical type 1 and type 2 IGF receptors, and 2) a secreted and membrane-associated small IGF-binding protein.