Evening chronotype, disordered eating behavior, and poor dietary habits in bipolar disorder.

OBJECTIVE: Our aim was to evaluate the relationship between evening chronotype, a proxy marker of circadian system dysfunction, and disordered eating behavior and poor dietary habits in individuals with bipolar disorder (BD). METHODS: In this cross-sectional study, we evaluated 783 adults with BD. Chronotype was determined using item 5 from the reduced Morningness-Eveningness Questionnaire. The Eating Disorder Diagnostic Scale (EDDS) and the Rapid Eating Assessment for Participants-Shortened Version (REAP-S) were used to assess disordered eating behavior and dietary habits respectively. General linear models and logistic regression models were utilized to evaluate differences between chronotype groups. RESULTS: Two hundred and eight (27%) BD participants self-identified as having evening chronotypes. Compared to non-evening types, evening types were younger (P < 0.01) and, after controlling for age, had higher mean EDDS composite z-scores (P < 0.01); higher rates of binge-eating (BE) behavior (P = 0.04), bulimia nervosa (P < 0.01), and nocturnal eating binges (P < 0.01); and a higher body mass index (P = 0.04). Compared to non-evening types, evening chronotypes had a lower REAP-S overall score (P < 0.01) and scored lower on the 'healthy foods' and 'avoidance of unhealthy food' factors. Evening types also skipped breakfast more often (P < 0.01), ate less fruit (P = 0.02) and vegetables (P = 0.04), and consumed more fried foods (P < 0.01), unhealthy snacks (P = 0.02), and soft drinks (P = 0.01). CONCLUSIONS: Our findings suggest that the circadian system plays a role in the disordered eating and unhealthy dietary behaviors observed in BD patients. The circadian system may therefore represent a therapeutic target in BD-associated morbidity that warrants further investigation.

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Identification of vibrational signatures from short chains of interlinked molecule-nanoparticle junctions obtained by inelastic electron tunnelling spectroscopy.

Short chains containing a series of metal-molecule-nanoparticle nanojunctions are a nano-material system with the potential to give electrical signatures close to those from single molecule experiments while enabling us to build portable devices on a chip. Inelastic electron tunnelling spectroscopy (IETS) measurements provide one of the most characteristic electrical signals of single and few molecules. In interlinked molecule-nanoparticle (NP) chains containing typically 5-7 molecules in a chain, the spectrum is expected to be a superposition of the vibrational signatures of individual molecules. We have established a stable and reproducible molecule-AuNP multi-junction by placing a few 1,8-octanedithiol (ODT) molecules onto a versatile and portable nanoparticle-nanoelectrode platform and measured for the first time vibrational molecular signatures at complex and coupled few-molecule-NP junctions. From quantum transport calculations, we model the IETS spectra and identify vibrational modes as well as the number of molecules contributing to the electron transport in the measured spectra.

Characteristics of the child behavior checklist in adolescents with depression associated with bipolar disorder.

BACKGROUND: The child behavior checklist-Juvenile bipolar disorder phenotype (CBCL-JBD) has been proposed as a distinct profile specific to children and adolescents who have been diagnosed with bipolar disorder. The objective of this study was to examine whether bipolar disorder youth with depression exhibit the "CBCL-Juvenile bipolar disorder phenotype." METHODS: Thirty-two adolescents, ages 12-18 years, with a depressive episode associated with bipolar I disorder were recruited, and their primary caregivers completed the CBCL. RESULTS: Only the internalizing subscale (mean=70.2, SD=9.7) and total score (mean=71.5, SD=8.9) reached clinical significance (>70). Moreover, the CBCL-JBD profile scores of our subjects (204.6, SD=27.5) did not reach clinical significance (>210). LIMITATIONS: Our subjects differed demographically from those in studies that have confirmed the CBCL-Juvenile bipolar disorder phenotype with regards to sex, age and ADHD comorbidity, thus limiting the interpretability of our comparisons with other studies. Furthermore, our investigation involved a small sample size and did not include a control group, which should be addressed in future studies. CONCLUSIONS: The results of our study suggest that the CBCL-JBD profile is not characteristic of depressed youth with bipolar disorder. Better assessment tools for making an accurate and efficient diagnosis of bipolar disorder are needed so that appropriate treatment can be implemented and significant morbidity and mortality are minimized.

Improved gas sensing activity in structurally defected bilayer graphene.

Graphene is a two-dimensional material with a capability of gas sensing, which is here shown to be drastically improved by inducing gentle disorder in the lattice. We report that by using a focused ion beam technique, controlled disorder can be introduced into the graphene structure through Ga(+) ion irradiation. This disorder leads to an increase in the electrical response of graphene to NO(2) gas molecules by a factor of three in an ambient environment (air). Ab initio density functional calculations indicate that NO(2) molecules bind strongly to Stone-Wales defects, where they modify electronic states close to the Fermi level, which in turn influence the transport properties. The demonstrated gas sensor, utilizing structurally defected graphene, shows faster response, higher conductivity changes and thus higher sensitivity to NO(2) as compared to pristine graphene.

The effect of mouthrinses on oral malodor: a systematic review.

OBJECTIVE: The objective of this study is to systematically review the literature regarding the impact of mouthrinses on oral malodor and present evidence for the treatment effects of mouthrinses on oral malodor. MATERIAL AND METHODS: PubMed-MEDLINE, the Cochrane-CENTRAL and EMBASE were searched through February 10, 2012 to identify appropriate studies. Volatile sulphur compound measurements, organoleptic measurements and tongue coating were selected as outcome variables. SEARCH RESULTS: The independent screenings of 333 unique titles and paper abstracts revealed 12 publications (12 experiments) that met the eligibility criteria. Means and standard deviations were extracted. The results were separated into short-term (<3 weeks) and longer-term (≥3 weeks) studies. CONCLUSION: In this review, nearly all mouthwashes with active ingredients had beneficial effects in reducing oral malodor in both short- and longer-term studies. The most compelling evidence was provided for chlorhexidine mouthwashes, and those that contained a combination of cetyl pyridinum chloride and zinc provided the best evidence profile on oral malodor. Little data with respect to tongue coating were available, and none of the studies showed a beneficial effect for this parameter.

Enabling measurements of low-conductance single molecules using gold nanoelectrodes.

A high resistance nanogap platform was used to trap and electrically characterize 30 nm thiolated double-stranded DNA molecules. High resolution scanning electron microscopy was also used to image the trapped DNA strands. It was found that the surface state of the electrodes and underlying substrate could influence the measurements of trapped molecules when the measured resistances were on the order of TΩ or greater. Hydrophilic surfaces gave rise to larger leakage currents that could potentially mask the underlying signals from molecules positioned in the nanogap. Finally, the careful handling of the samples and control of the environment is essential to avoid surface charging of the oxide substrate layer as these parasitic charges affect electrical measurements of the nanogap. The presented results thus outline some important considerations when making low-conductance measurements on molecules and should prove useful for the characterization of molecules in molecular electronics or sensors employing nanogap platforms.

The age-dependent effects of selective serotonin reuptake inhibitors in humans and rodents: A review.

The selective serotonin reuptake inhibitor (SSRI) Prozac® (fluoxetine) is widely prescribed for the treatment of depression and anxiety-related disorders. While extensive research has established that fluoxetine is safe for adults, safety is not guaranteed for (unborn) children and adolescents. Some clinical studies have reported adverse outcomes, such as premature birth, neonatal cardiovascular abnormalities, and pulmonary hypertension in children whose mothers used SSRIs during pregnancy. In addition, several reports show that adolescent fluoxetine treatment increases risk for suicidal behavior. Despite these studies, fluoxetine is not contraindicated in the treatment of depressed pregnant women and adolescents. Longitudinal research in humans is limited because of ethical reasons and time constraints, and to overcome these limitations, rodents are used to increase insight in the age-dependent effects of fluoxetine exposure. It has been established that neonatal and adolescent fluoxetine exposure leads to paradoxical anxiety- and depression-like features in later life of rats and mice, although in some studies adolescent fluoxetine exposure was without effects. These age-dependent outcomes of fluoxetine may be explained by serotonin's neurotrophic effects, which may vary according to the developmental stage of the brain due to epigenetic modifications. Here we review the existing evidence for the age-dependent effects of fluoxetine in humans and rodents, address the gaps in our current knowledge and propose directions for future research. Given the overlap between human and rodent findings, rodents provide heuristic value in further research on the age-dependent effects of SSRIs.

Assessment of a nanoparticle bridge platform for molecular electronics measurements.

A combination of electron beam lithography, photolithography and focused ion beam milling was used to create a nanogap platform, which was bridged by gold nanoparticles in order to make electrical measurements and assess the platform under ambient conditions. Non-functionalized electrodes were tested to determine the intrinsic response of the platform and it was found that creating devices in ambient conditions requires careful cleaning and awareness of the contributions contaminants may make to measurements. The platform was then used to make measurements on octanethiol (OT) and biphenyldithiol (BPDT) molecules by functionalizing the nanoelectrodes with the molecules prior to bridging the nanogap with nanoparticles. Measurements on OT show that it is possible to make measurements on relatively small numbers of molecules, but that a large variation in response can be expected when one of the metal-molecule junctions is physisorbed, which was partially explained by attachment of OT molecules to different sites on the surface of the Au electrode using a density functional theory calculation. On the other hand, when dealing with BPDT, high yields for device creation are very difficult to achieve under ambient conditions. Significant hysteresis in the I-V curves of BPDT was also observed, which was attributed primarily to voltage induced changes at the interface between the molecule and the metal.

Sphingosine kinase as an oncogene: autocrine sphingosine 1-phosphate modulates ML-1 thyroid carcinoma cell migration by a mechanism dependent on protein kinase C-alpha and ERK1/2.

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading.

Here we used electrospray ionization mass spectrometry for quantitative determination of lipid molecular species in human fibroblasts and their plasma membrane incorporated into enveloped viruses. Both influenza virus selecting ordered domains and vesicular stomatitis virus (VSV) depleted of such domains [Scheiffele, P., et al. (1999) J. Biol. Chem. 274, 2038-2044] were analyzed. The major difference between influenza and VSV was found to be a marked enrichment of glycosphingolipids in the former. The effect of chronic cholesterol loading on viral lipid composition was studied in Niemann-Pick type C (NPC) fibroblasts. Both NPC-derived influenza and VSV virions contained increased amounts of cholesterol. Furthermore, polyunsaturated phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were enriched in NPC-derived virions at the expense of the monounsaturated ones. When normal fibroblasts were acutely loaded with cholesterol using cyclodextrin complexes, an adjustment toward increasingly unsaturated phospholipid species was observed, most clearly for phosphatidylcholine and sphingomyelin. Our results provide evidence that (1) glycosphingolipids are enriched in domains through which influenza virus buds, (2) chronic cholesterol accumulation increases the cholesterol content of both glycosphingolipid-enriched and intervening plasma membrane domains, and (3) an increase in membrane cholesterol content is accompanied by an increased level of polyunsaturated species of the major membrane phospholipids. We suggest that remodeling of phospholipids toward higher unsaturation may serve as both an acute and a long-term adaptive mechanism in human cellular membranes against cholesterol excess.

Sixty thousand woman-years of experience on the levonorgestrel intrauterine system: an epidemiological survey in Finland.

OBJECTIVE: An epidemiological survey was carried out in Finland to establish the performance of the levonorgestrel intrauterine system (LNG-IUS) in a large population of women regularly using the system for contraception. STUDY DESIGN: All women who had LNG-IUS inserted between 1990 and 1993 were sent a questionnaire with questions about general health status, reproductive and contraceptive history and gynecological problems and a set of questions about their experience on the LNG-IUS. RESULTS: The women who had a LNG-IUS inserted between April 1990 and December 1993 were asked to participate in an epidemiological survey. At the insertion visit, the women were asked to fill in a questionnaire, to consent to further use of the information by signing their initials and to return the questionnaire to the manufacturer. The number of returned forms was 23,885. The names and addresses of these women were acquired from the National Register and a questionnaire with 75 questions was sent to them. Of the questionnaires, 75% were returned. Experience covered 58,600 woman-years. The mean age of the users of the LNG-IUS was 40 years. Most of them (99.3%) were parous, most often with two or three children. The cumulative 5-year Pearl index was 0.18 and there were 108 pregnancies in the study population during the use of the LNG-IUS. The continuation rates for 1, 2, 3, 4 and 5 years were 0.94, 0.87, 0.82, 0.76 and 0.65, respectively. Removal before the full 5 years was most common among the youngest age group. Bleeding disorders, infections and pain during LNG-IUS use were associated with the highest risk for discontinuation. The risk of premature removal was markedly diminished among women with totally or occasionally missed periods. CONCLUSIONS: The response rate to the survey was high and the sample studied was exceptionally large. The continuation rate of LNG-IUS was high up to 5 years. The symptoms associated with premature removal agreed with results from earlier randomized studies of the LNG-IUS.

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Sixty thousand woman-years of experience on the levonorgestrel intrauterine system: an epidemiological survey in Finland.

OBJECTIVE: An epidemiological survey was carried out in Finland to establish the performance of the levonorgestrel intrauterine system (LNG-IUS) in a large population of women regularly using the system for contraception. STUDY DESIGN: All women who had LNG-IUS inserted between 1990 and 1993 were sent a questionnaire with questions about general health status, reproductive and contraceptive history and gynecological problems and a set of questions about their experience on the LNG-IUS. RESULTS: The women who had a LNG-IUS inserted between April 1990 and December 1993 were asked to participate in an epidemiological survey. At the insertion visit, the women were asked to fill in a questionnaire, to consent to further use of the information by signing their initials and to return the questionnaire to the manufacturer. The number of returned forms was 23,885. The names and addresses of these women were acquired from the National Register and a questionnaire with 75 questions was sent to them. Of the questionnaires, 75% were returned. Experience covered 58,600 woman-years. The mean age of the users of the LNG-IUS was 40 years. Most of them (99.3%) were parous, most often with two or three children. The cumulative 5-year Pearl index was 0.18 and there were 108 pregnancies in the study population during the use of the LNG-IUS. The continuation rates for 1, 2, 3, 4 and 5 years were 0.94, 0.87, 0.82, 0.76 and 0.65, respectively. Removal before the full 5 years was most common among the youngest age group. Bleeding disorders, infections and pain during LNG-IUS use were associated with the highest risk for discontinuation. The risk of premature removal was markedly diminished among women with totally or occasionally missed periods. CONCLUSIONS: The response rate to the survey was high and the sample studied was exceptionally large. The continuation rate of LNG-IUS was high up to 5 years. The symptoms associated with premature removal agreed with results from earlier randomized studies of the LNG-IUS.

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

Length of use and symptoms associated with premature removal of the levonorgestrel intrauterine system: a nation-wide study of 17,360 users.

OBJECTIVE: To establish the continuation rates of the levonorgestrel intrauterine system (LNG IUS) and symptoms associated with its premature removal. SAMPLE AND SETTING: All women in Finland who had a LNG IUS inserted between April 1990 and December 1993 and whose doctor had filled in and returned a form at the insertion visit. This study population consists of 46% of all the LNG IUSs sold in Finland between 1990 and 1993. DESIGN: A questionnaire on reproductive and contraceptive history, gynaecological problems and symptoms experienced during the use of the LNG IUS was sent to 23,885 LNG IUS users. A total of 17,914 questionnaires were returned (response rate 75%). The results cover experience from 58,600 woman years. A log-rank-test was used to test differences in continuation rates. Multivariate analyse were performed using Cox's proportional hazard model. RESULTS: The LNG IUS was prematurely removed from 5175 women. The one, two, three, four and five year continuation rates were 93%, 87%, 81%, 75% and 65%, respectively. The symptoms during the use of the LNG IUS most strongly associated with its premature removal were excessive bleeding and spotting, and infections and pain. The risk of premature removal was markedly lower among women who had occasional or total absence of menstruation. Premature removal was less likely in the oldest age group. CONCLUSIONS: The continuation rate of the LNG IUS compares favourably with other long-acting contraceptive systems. Totally or occasionally absent menstruation was strongly associated with prolonged continuation.

Characterization of a human basophil-like cell line (LAMA-84).

LAMA-84, a human leucocytic cell line, which upon establishment was described as having megakaryocytic, erythroid and granulocytic characteristics, was analysed for expression of various differentiation markers. In addition to some of the previously described phenotypic characteristics, this cell line was found to express mRNA for several proteins characteristic for basophilic leucocytes and mast cells. The authors show that LAMA-84 cells express mRNA for the mast cell tryptase, the proteoglycan core protein, carboxypeptidase A and the alpha and beta chains of the high affinity IgE receptor (Fc epsilon RI). The authors examined the potential of LAMA-84 to differentiate in serum-free medium or after DMSO or PMA treatment. Depending on the inducing factor, surface expression of the Fc epsilon RI alpha-chain was increased from 20% to 35-50% of the cells and mRNA levels for tryptase were increased in serum-free medium and after DMSO treatment. LAMA-84 was found to express CD13, CDw17, CD29, CD33, CD40, CD45 and CD117. Furthermore, mRNA for the eosinophil/basophil markers Charcot-Leyden crystal (CLC) protein and the major basic protein (MBP), as well as the erythrocyte differentiation marker alpha-globin, was detected. However, the authors observed only trace amounts of mRNA for another erythroid differentiation marker (glycophorin), trace amounts of the megakaryocytic marker GPIIIa, and no detectable level of GPIb alpha. By comparing the expression pattern of a panel of differentiation markers in LAMA-84, and a second human cell line (KU812) expressing a basophil phenotype, it is evident that these cell lines, which presently are the only two cell lines identified with basophilic characteristics, share a large number of phenotypic characteristics.

Stem cell factor-dependent human cord blood derived mast cells express alpha- and beta-tryptase, heparin and chondroitin sulphate.

The present study sought to determine the expression of alpha- and beta-tryptase in in vitro differentiated human cord blood derived mast cells. We also analysed the glycosaminoglycan composition and the phenotype of the cells. The major protease in human mast cells is tryptase, and cDNAs for two different human tryptases have been characterized, the so-called alpha- and beta-tryptase. By reverse transcriptase-polymerase chain reaction (RT-PCR) we could show that stem cell factor (SCF)-dependent cord blood derived mast cells express both alpha- and beta-tryptase. Furthermore, the cells were stained with a monoclonal antibody (mAb) against tryptase, and the tryptase was enzymatically active cleaving the substrate Z-Gly-Pro-Arg- methoxy-2- naphthylamide (MNA). The majority of the cord blood derived mast cells could also be stained with mAbs against chymase, cathepsin G and CD68. They also expressed Kit/SCFR (CD117), CD13, CD29 and CD45 on the cell surface. The proteoglycan-derived polysaccharide composition of the cells was estimated to be 25-35% of heparin origin and 65-75% of chondroitin sulphate origin. Hence, the cord blood derived mast cells exhibit a phenotype in common with the so-called MCTC type of human mast cells.

Phenotypic characterization of the human mast-cell line HMC-1.

The cell line HMC-1, derived from a patient with mast cell leukaemia, is the only established cell line exhibiting a phenotype similar to that of human mast cells. This paper reports on a detailed characterization of the expression of a panel of markers for various types of immature and mature haematopoietic cells in the HMC-1. We also studied the potential of HMC-1 to differentiate upon treatment with conditioned media from the human T-cell line Mo, retinoic acid or DMSO. HMC-1 was found to express several mast cell-related markers. A high expression of Kit, the receptor for stem-cell factor, was detected. The majority of the cells were stained with a MoAb against the mast cell-specific serine protease tryptase. Of particular interest was the finding that beta-tryptase mRNA, but not alpha-tryptase mRNA, was expressed in HMC-1. Using enzyme-histochemistry we were able to show that the beta-tryptase was enzymatically active, indicating that tryptase can form active homotetramers. Both heparin and chondroitin sulfate were found to be present in approximately equal amounts. HMC-1 lacked surface expression of the high-affinity IgE receptor, which was confirmed by the absence of mRNA of the alpha- and beta-chains of the IgE-receptor complex. However, a strong expression of the gamma-chain of the IgE-receptor complex was detected. A positive staining of the monocyte/macrophage marker CD68 was obtained, as well as a strong hybridization signal for the eosinophilic/basophilic-related differentiation marker the Charcot-Leyden crystal. Treatment of HMC-1 with conditioned media from the human T-cell line Mo, retinoic acid or DMSO induced only moderate changes in the surface or intracellular expression of the studied markers. The agents tested neither induced any of the monocyte/granulocyte markers examined, nor expression of the Fc epsilon RI alpha-chain.

Characterization of a tryptase mRNA expressed in the human basophil cell line KU812.

The expression of a tryptic serine protease was detected in the cell line KU812 by Northern blot analysis with an oligonucleotide probe directed against a conserved region present in all of the five presently cloned human mast cell tryptases. PCR primers designed for the amplification of a nearly full-length copy of tryptase mRNAs were used to study the identity of the KU812 tryptase. Ten clones were characterized and all were found to be identical to one of the tryptases previously cloned from a human skin cDNA library. This tryptase has been thought to originate from mast cells of the skin. Two possible explanations may account for the observed identity between the presumed mast cell tryptase and the KU812 tryptase. Firstly, it is possible that the KU812 tryptase is a basophil-specific tryptase which has previously been cloned from a human skin cDNA library containing low levels of cDNA copies derived from basophils in the starting material. Secondly, the KU812 cell line, and possibly normal basophils, express a tryptase which is identical to one of the tryptases expressed in normal skin mast cells. We cannot at present rule out any of the two possibilities, but we favour the second explanation as being the most likely.