Triglycerides cross the blood-brain barrier and induce central leptin and insulin receptor resistance.

OBJECTIVE: Resistance at the brain receptors for leptin and insulin has been associated with increased feeding, obesity and cognitive impairments. The causal agent for central resistance is unknown but could be derived from the blood. Here we postulate whether hypertriglyceridemia, the major dyslipidemia of the metabolic syndrome, could underlie central leptin and insulin resistance. DESIGN: We used radioactively labeled triglycerides to measure blood-brain barrier (BBB) penetration, western blots to measure receptor activation, and feeding and cognitive tests to assess behavioral endpoints. RESULTS: Human CSF was determined to contain triglycerides, a finding previously unclear. The radioactive triglyceride triolein readily crossed the BBB and centrally administered triolein and peripherally administered lipids induced in vivo leptin and/or insulin resistance at hypothalamic receptors. Central triolein blocked the satiety effect of centrally administered leptin. Decreasing serum triglycerides with gemfibrozil improved both learning and memory inversely proportionate to triglyceride levels. CONCLUSIONS: Triglycerides cross the blood-brain barrier rapidly, are found in human cerebrospinal fluid, and induce central leptin and insulin receptor resistance, decreasing satiety and cognition.

Ozonation of estrogenic chemicals in biologically treated sewage.

The present study shows that ozonation of effluents from municipal wastewater treatment plants (WWTPs) is likely to be a future treatment solution to remove estrogens and xeno-estrogens. The required ozone dose and electrical energy for producing the ozone were determined in two WWTP effluents for removal of 17 estrogenic chemicals. The estrogenic compounds included parabens, industrial phenols, sunscreen chemicals, and steroid estrogens. The obtained values of Electrical Energy per Order (EEOs) for the treatment of the estrogens were in the range 0.14-1.1 kWh/m(3) corresponding to 1.7-14 g O3/m(3). It is furthermore suggested that UV-absorbance is a useful parameter for online control of the ozone dose in a full scale application since the absorbance of the WWTP effluents and the remaining concentration of the estrogens and xeno-estrogens correlated well with the applied ozone dose.

Bioassay of prostate-specific antigen (PSA) using microcantilevers.

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.

Rat mesangial cells express two unique isoforms of laminin which modulate mesangial cell phenotype.

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.

Gynecologic specimen analysis by multiangle light scattering in a flow system.

A flow-system instrument is described in which the laser light scattered by a mammalian cell is sampled simultaneously at up to 32 angles between 0 degrees and 21 degrees from the laser beam axis as the cell passes through the beam. The scatter pattern for each cell is stored by a computer for later analysis. Various data-processing techniques are discussed. Results of preliminary application of the instrument to the analysis of normal and abnormal gynecologic specimens are presented.

Loxapine versus perphenazine in psychotic patients. A double-blind, randomized, multicentre trial.

A double-blind, randomized, multicentre trial was carried out in 47 psychotic patients to evaluate the efficacy of oral treatment with loxapine compared with perphenazine. In total, 22 patients were included in diagnostic Group I (cases of acute schizophrenia and psychogenic (reactive) psychoses). The average maximum daily dose was 60.0 mg in the loxapine group and 36.8 mg in the perphenazine group. After 3-weeks' treatment, no significant differences were found between the two treatment groups according to the Brief Psychiatric Rating Scale (BPRS), Clinical Global Impression (CGI) Scale or side-effect records. Twenty-five patients were included in diagnostic Group II (cases of chronic schizophrenia). The average daily dosage was 81.1 mg in the loxapine group and 90.1 mg in the perphenazine group. After 10 to 12-weeks' treatment, no significant differences between the two treatment groups could be found according to BPRS, CGI scale, Nurses' Observation Scale for In-patient Evaluation (NOSIE) or side-effect records. The diastolic blood pressure (lying and standing) tended to increase slightly in both treatment groups. In conclusion, it was found that loxapine and perphenazine seemed to be equally effective and, based on experience with parenteral loxapine treatment, it is suggested that further investigation of oral loxapine should be carried out in psychotic patients in whom agitation is a feature.

Effects of temperature on development of Onchocerca volvulus in Simulium ochraceum, and longevity of the simuliid vector.

The effects of temperature on the development of Onchocerca volvulus in Simulium ochraceum, and on the vector's longevity were studied under simulated environmental conditions in the laboratory. When S. ochraceum (which had fed on a person infected with O. volvulus) were maintained at different, constant temperatures (10-30 C), larvae developed to the infective stage between 20 and 28 C. The time required for development to the infective stage depended on the relationship of y = -0.3760 + 0.0222x (y = velocity in development; x = rearing temperature). However, larval development to the infective stage occurred in all groups kept at different temperatures by day and by night (25 C during the day and 10-18 C during the night), although the developmental period was prolonged (10 days in the group held at 25/18 C, and 13 days in other groups). The percentage of females harboring infective larva(e) among those maintained was highest at a constant temperature of 22 C (21.9%), followed by 15.0% at 25 C. When S. ochraceum females were held at day/night differing temperatures, much higher rates were observed at 25/16 C (30.0%) and at 25/14 C (23.0%). Our results suggest that the distribution of onchocerciasis in Guatemala may be related to ambient temperature and to day/night temperature cycles.

Enzyme assay for identification of pectin and pectin derivatives, based on recombinant pectate lyase.

A simple method was developed for fast identification of pectin, based on a recombinant endopectate lyase cloned from Aspergillus niger. When pectin was demethylated and treated with pectate lyase, beta-elimination occurred, resulting in a double bond between C-4 and C-5 in the galacturonic acid residue of the released nonreducing end. The formation of double bonds produced an increase in light absorption, which was detected at 235 nm. The assay was tested on pectin of different origins (apple, orange, sugar beet, sunflower, celery, lemon), pectin derivatives (amidated pectin), and speciality types such as low molecular weight and low %DE (degree of esterification, percentage of galacturonic acid groups esterified with methanol) pectin. The highest response was given by pectate (pectin with %DE< 5) and the lowest by pectin extracted from sugar beet. No other gums (carboxymethylcellulose, carrageenan, locust bean gum, tragacanth, gellan, tamarind, xanthan, amylogum, sodium alginate, or agar) gave any response. Members of IPPA (International Pectin Producers Association) have evaluated the validity of the assay in a ring test. All members of the Association were able to identify pectin from other gums in a blind test. The method can replace more laborious and ambiguous identification tests which exist today.

[Did the distribution of trauma treatment by general practitioners and emergency departments in the county of Ringkøbing change after the introduction of the on-call coverage for general practitioners?]. / Aendres fordelingen af skadebehandlingen i Ringkøbing Amt mellem almen praksis og skadestue ved etablering af storvagtkredse for alment praktiserende laeger?

The aim of the study was to investigate the changes in minor trauma treatment structure after a reduction in the number of general practitioners on call in a county, where minor trauma treatment is supposed to be carried out by general practitioners and only major trauma is supposed to be treated at the hospital Accident and Emergency Department. The design was a cross-sectional analysis of trauma treatment in Ringkøbing County before and after the reduction in the number of general practitioners on call. Over a four week period before and after the reduction in the number of general practitioners on call all trauma treatment at the Accident and Emergency Departments was registered together with trauma treatment by general practitioners. Furthermore a questionnaire was given before and after the reduction to a sample of the population regarding the population's behaviour and attitude towards minor trauma. Analysis showed that there was a minor reduction in the total number of trauma treatments after the reduction in the number of general practitioners on call was made. The percentage of patients that were treated at the Accident and Emergency Departments at hospital directly without being referred from general practitioners was reduced from 30% to 21%. The population's behaviour and attitude towards minor trauma was unchanged.

Abnormal development of glomerular endothelial and mesangial cells in mice with targeted disruption of the lama3 gene.

Mice with targeted disruption of the lama3 gene, which encodes the alpha3 chain of laminin-5 (alpha3beta3gamma2, 332), develop a blistering skin disease similar to junctional epidermolysis bullosa in humans. These animals also develop abnormalities in glomerulogenesis. In both wild-type and mutant animals (lama3(-/-)), podocytes secrete glomerular basement membrane and develop foot processes. Endothelial cells migrate into this scaffolding and secrete a layer of basement membrane that fuses with the one formed by the podocyte. In lama3(-/-) animals, glomerular maturation arrests at this stage. Endothelial cells do not attenuate, develop fenestrae, or form typical lumens, and mesangial cells (MCs) were not identified. LN alpha3 subunit (LAMA3) protein was identified in the basement membrane adjacent to glomerular endothelial cells (GEnCs) in normal rats and mice. In developing rat glomeruli, the LAMA3 subunit was first detectable in the early capillary loop stage, which corresponds to the stage at which maturation arrest was observed in the mutant mice. Lama3 mRNA and protein were identified in isolated rat and mouse glomeruli and cultured rat GEnCs, but not MC. These data document expression of LAMA3 in glomeruli and support a critical role for it in GEnC differentiation. Furthermore, LAMA3 chain expression and/or another product of endothelial cells are required for MC migration into the developing glomerulus.

Chromosome investigation of Danish A. I. beef bulls.

In 1973 all Danish A.I. beef bulls were tested by chromosome analysis, in order to eliminate bulls with Robertsonian translocation (centric fusion translocations). From abroad it has been reported that this aberration reduced fertility. Of 65 bulls none was affected. The literature concerning chromosome investigation of populations of A.I. bulls are summarized. It is proposed that all imported bulls should be tested by chromosome banding methods, before the bulls are used.

Linkage analysis of loci controlling blood groups and the rectovaginal constriction syndrome in Jersey cattle.

The possibility of linkage between the recessive gene controlling the rectovaginal constriction (RVC) syndrome in Jersey cattle and 13 loci controlling blood groups and polymorphic proteins was studied. No evidence of close to moderate linkage was found between the RVC locus and any of the systems A, B, C, F, L, S, Z, R', Hb, Tf, Am-1 and Ca. No definite conclusion was possible with the M system.

Characterization of a Cystine-Rich Polyphenolic Protein Family from the Blue Mussel Mytilus edulis L.

Marine bivalve mollusks synthesize, in the phenol and accessory glands of the foot, proteins that integrate the post-translationally hydroxylated amino acid 3,4-dihydroxyphenylalanine (DOPA) into their primary sequence. These polyphenolic proteins serve as structural and adhesive components of the byssal threads which form the extraorganismic holdfast. One family of byssal precursors, previously characterized in a number of mytiloid species, consists of proteins between 70-130 kDa containing 8-18 mol % DOPA. The high molecular weight precursor isolated from the foot of the blue mussel (Mytilus edulis Linnaeus, 1758) is here designated as Mefp-1. We now present evidence for the occurrence, in M. edulis, of a second, structurally unrelated, family of DOPA proteins (Mefp-2) of about 42-47 kDa. These novel proteins contain 2-3 mol % DOPA and, in startling contrast to Mefp-1, are also enriched in the disulphide-containing amino acid cystine (6-7 mol %). Consideration of the amino acid compositions of Mefp-1 and 2 and of the terminal adhesive plaques of byssal threads suggests that Mefp-2 makes up about 25% of plaque protein, whereas Mefp-1 content is about 5%. The Mefp-2 family exhibits electrophoretic microheterogeneity, but members share similar N- and C-terminal amino acid sequences. Analysis of peptides isolated after tryptic hydrolysis suggests that the primary sequence of Mefp-2 is tandemly repetitive, with at least three types of motif. The sequence degeneracy of the motifs is greater than in Mefp-1. Mefp-2 has minimal sequence homology with known structural proteins and may be a structural element of the plaque matrix.

Cadmium-induced metallothionein expression during embryonic and early larval development of the mollusc Crassostrea virginica.

Newly fertilized eggs of the oyster Crassostrea virginica were exposed to 0.2 microM Cd and sampled during the first 24 hr of embryonic and larval development for determination of the temporal patterns of total bioaccumulated and metallothionein (MT)-bound Cd concentrations and the concentrations of MT mRNA. In comparison with controls, exposure to this concentration of Cd resulted in delayed development to the D-stage veliger larval stage. Maternal MT mRNA, which was carried over into the egg, declined immediately after fertilization. In controls, levels of MT mRNA were stable after this initial decline, and little or no detectable Cd was bound to MTs. Accumulation of new MT mRNA was observed in the exposed group after 12 hr of exposure when increases due to induction by Cd had occurred. Total accumulated Cd concentrations, which ranged from 0.04 to 0.1 pmol Cd/microg DNA in unexposed individuals, increased to 13.5 pmol Cd/microg DNA after 24 hr in Cd-exposed individuals. There was an increase in MT-bound Cd in exposed individuals prior to MT induction, which may be explained in part as Cd binding to maternal MT. This was detected as binding to CvNAcMT, the expected N-acetylated form of the oyster MT. Amounts of Cd bound to this form of MT increased, thereafter, and was followed by increases in binding to CvMT, the unexpected, nonacetylated form of the oyster MT, which has been associated with Cd toxicity, possibly in relation to the effects of Cd on normal cotranslational processing.

Cadmium-induced expression of metallothionein and suppression of RNA to DNA ratios during molluscan development.

One-week-old larvae of the mollusc Crassostrea virginica, an oyster, were exposed to Cd concentrations ranging from the control with no added Cd to 0.2 microM CdCl2 for 24 hr. Concentration-dependent increases in total cadmium accumulation, cytosolic and metallothionein-bound Cd concentrations, and levels of the MT mRNA were detectable over this range of exposure concentrations. Increases in these measures were apparent at the lowest exposure concentration of 0.005 microM, with dramatic increases observed between 0.04 and 0.2 microM Cd. Although the concentrations of MT-bound Cd increased, the fraction of total Cd that was bound to MTs declined in larvae treated with 0.04 and 0.2 microM Cd, presumably due to the inability of MT biosynthesis to keep up with the higher rates of Cd uptake at the higher concentrations. This decreased effectiveness of MTs in sequestering Cd in relation to the total Cd load and the associated increase in the nonthionein Cd fraction at these higher treatment conditions coincided with a decline in the RNA to DNA ratio and an increase in a nonacetylated variant of the MT. The former is indicative of an inhibition of larval metabolism and growth. The latter is an unexpected form for this MT and is believed to signify disruption of normal cotranslational modification of the N-terminal amino acid sequence.

Cantilever-based optical deflection assay for discrimination of DNA single-nucleotide mismatches.

Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.

A flow-system multiangle light-scattering instrument for cell characterization.

A flow-system cell-analysis instrument is described in which cells from a heterogeneous population are characterized by their light-scatter patterns alone. As the cells pass at high speed through a focused helium/neon laser beam, the scatter pattern from each cell is sampled simultaneously at up to 32 angles between 0 degrees and 30 degrees with respect to the laser beam axis, and the scatter pattern for each cell is transferred to a computer. A mathematical clustering algorithm is used to determine the number of classes into which the cells can be divided, and a linear separation algorithm is used to find the boundaries between the classes. Preliminary results on exfoliated cells from gynecological specimens are presented. This technique may be useful for automated prescreening of gynecological specimens.