Monoclonal antibodies to bovine milk lipoprotein lipase. Evidence for proteolytic degradation of the native enzyme

Lipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose. Polyacrylamide gel electrophoresis showed a single protein with an apparent molecular weight of 55,000 in the trailing edge of the elution profile; fractions in the leading edge contained additional proteins with molecular weights of 36,000 and 18,000-22,000. Nine monoclonal antibodies were prepared against the 55,000-dalton protein. By immunoblotting, we show that the Mr = 18,000-22,000 components share common antigen determinants with the 55,000-dalton protein, suggesting that they represent proteolytic degradation products. Incubation of partially purified lipoprotein lipase for 24 h at 37 degrees C results in breakdown of the 55,000-dalton protein with concomitant enrichment in lower Mr components; the proteolytic activity is prevented by incubating the milk with phenylmethane, sulfonyl fluoride prior to chromatography on heparin-Sepharose. This study shows the presence of milk proteases which co-purify and degrade lipoprotein lipase. We suggest that this degradation could account for part of the known instability of the enzyme.     

Ecosystem changes associated with grazing in subhumid South American grasslands

Question: What are the changes in vegetation structure, soil attributes and mesofauna associated with grazing in mesic grasslands? Location: Southern Campos of the Río de la Plata grasslands, in south‐central Uruguay. Methods: We surveyed seven continuously grazed and ungrazed paired plots. Plant and litter cover were recorded on three 5‐m interception lines placed parallel to the fence in each plot. We extracted soil fauna from a 10 cm deep composite sample and analysed the oribatids. Soil attributes included bulk density, water content, organic carbon (in particulate and mineral associated organic matter) and nitrogen content and root biomass at different depths. Changes in floristic, Plant Functional Types and mesofauna composition were analysed by Non‐metric Multidimensional Scaling. Results: Species number was lower in ungrazed than in grazed plots. Of 105 species in grazed plots only three were exotics. Shrub and litter cover were significantly higher inside the exclosures, while the cover of Cyperaceae‐Juncaceae was lower. Grazing treatments differed significantly in plant and oribatid species composition. Grazing exclusion significantly reduced soil bulk density and increased soil water content. Carbon content in particulate organic matter was lower in the upper soil of ungrazed sites, but deeper in the profile, grazing exclosures had 8% more carbon in the mineral associated organic matter. Conclusions Our results generally agree with previous studies but deviate from the results of previous analyses in (1) the increase of shrub cover in ungrazed sites; (2) the redistribution of the soil organic carbon in the profile and (3) the low invasibility of the prairies regardless of grazing regime.     

A multi-scale comparison of trait linkages to environmental and spatial variables in fish communities across a large freshwater lake

Species present in communities are affected by the prevailing environmental conditions, and the traits that these species display may be sensitive indicators of community responses to environmental change. However, interpretation of community responses may be confounded by environmental variation at different spatial scales. Using a hierarchical approach, we assessed the spatial and temporal variation of traits in coastal fish communities in Lake Huron over a 5-year time period (2001–2005) in response to biotic and abiotic environmental factors. The association of environmental and spatial variables with trophic, life-history, and thermal traits at two spatial scales (regional basin-scale, local site-scale) was quantified using multivariate statistics and variation partitioning. We defined these two scales (regional, local) on which to measure variation and then applied this measurement framework identically in all 5 study years. With this framework, we found that there was no change in the spatial scales of fish community traits over the course of the study, although there were small inter-annual shifts in the importance of regional basin- and local site-scale variables in determining community trait composition (e.g., life-history, trophic, and thermal). The overriding effects of regional-scale variables may be related to inter-annual variation in average summer temperature. Additionally, drivers of fish community traits were highly variable among study years, with some years dominated by environmental variation and others dominated by spatially structured variation. The influence of spatial factors on trait composition was dynamic, which suggests that spatial patterns in fish communities over large landscapes are transient. Air temperature and vegetation were significant variables in most years, underscoring the importance of future climate change and shoreline development as drivers of fish community structure. Overall, a trait-based hierarchical framework may be a useful conservation tool, as it highlights the multi-scaled interactive effect of variables over a large landscape.     

HISTOLOGICAL CHANGES DURING THE DEVELOPMENT OF THE BOVINE NUCHAL LIGAMENT

The histological changes occurring during the development of the bovine nuchal ligament have been observed in sections of formalin-fixed material from 21 animals ranging in age from 110 days of gestation to 10 yr. The elastic fibers which constitute the bulk of the adult ligament were initially few in number. During fetal development, the fibers showed a rapid increase both in number and in their stainability with the usual elastic stains. The average diameter of these elastic fibers increased only slowly until the last uterine month, at which time it began to increase very rapidly. This rapid rate of increase continued through the first 6 postnatal months, after which the rate of increase slowed markedly. However, the fiber diameter continued to rise steadily throughout the period of the study. During the fetal stage of development, the fibroblastic cells of the ligament exhibited unusual nuclear appearances which distinguish them from other fibroblasts. These consisted of marked clumping of the chromatin and an associated nuclear vacuolation or vesiculation. While these changes seem likely to be artefacts of fixation, their temporal correlation with elastin deposition and their demonstration in other tissue cells engaged in elastin production suggest that the factors responsible for these appearances may be related to elastin synthesis.     

Determination of twin zygosity: a comparison of DNA with various questionnaire indices.

This study examined cross-validation and test-retest reliability of questions and questionnaire indices commonly used for twin zygosity classification. Mothers of 58 monozygotic (MZ) and 52 dizygotic (DZ) same sex twin pairs were interviewed by telephone to answer questions regarding the similarity of their twins (mean age = 14.6 +/- 2.8 years). A logistic regression equation correctly classified 91% of both MZ and DZ twin pairs in our sample using 7 of the 12 zygosity questions. The internal consistency for the total questionnaire (Cronbach's alpha) was 0.88. The median two month temporal stability estimate for the individual questions was r = .56 and r = .79 for the test total. For the cross-validation, zygosity classification indices taken from 9 previous studies were applied to our sample and compared to classification according to DNA microsatellite analyses (agreement range = 44 to 100%). The accuracy of the classification indices was significantly lower than the original studies for 62% of the comparisons. If zygosity determination with DNA markers or blood group typing for all subjects is not feasible, rather than using classification indices based on other studies, an optimal classification scheme can be achieved by using a zygosity questionnaire of which the reliability and validity of the questions is established in a random subsample of the same twin cohort.     

G protein-coupled receptors control NMDARs and metaplasticity in the hippocampus

Long-term potentiation (LTP) and long-term depression (LTD) are the major forms of functional synaptic plasticity observed at CA1 synapses of the hippocampus. The balance between LTP and LTD or "metaplasticity" is controlled by G-protein coupled receptors (GPCRs) whose signal pathways target the N-methyl-D-asparate (NMDA) subtype of excitatory glutamate receptor. We discuss the protein kinase signal cascades stimulated by Galphaq and Galphas coupled GPCRs and describe how control of NMDAR activity shifts the threshold for the induction of LTP.     

Synthesis and Pharmacology of the enantiomers of the potential atypical antipsychotic agents 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT.

The optically pure enantiomers of the potential atypical antipsychotic agents 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 5) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}t etralin [5-OMe-(2,6-di-OMe)-BPAT, 6] were synthesized and evaluated for their in vitro binding affinities at alpha1-, alpha2-, and beta-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-5 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for alpha1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1000 nM) for the other receptor subtypes. (S)- and (R)-6 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-5, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereospecific, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 5 and 6. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 5 behaved as full serotonin 5-HT1A receptor agonists in this assay, while both enantiomers of 6 behaved as weak partial agonists. The potential antipsychotic properties of (S)- and (R)-5 were evaluated by establishing their ability to inhibit d-amphetamine-induced locomotor activity in rats, while their propensity to induce extrapyramidal side-effects (EPS) in man was evaluated by determining their ability to induce catalepsy in rats. Whereas (R)-5 was capable of blocking d-amphetamine-induced locomotor activity, indicative of dopamine D2 receptor antagonism, (S)-5 even enhanced the effect of d-amphetamine, suggesting that this compound has dopamine D2 receptor-stimulating properties. Since both enantiomers also were devoid of cataleptogenic activity, they are interesting candidates for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.     

Anti-inflammatory and redox-protective activities of citronellal

The anti-inflammatory and redox protective effects of the citronellal (CT) were evaluated using in vivo and in vitro tests. Intraperitoneal (i.p.) administration of CT (50, 100, and 200 mg/kg) inhibited (p < 0.05) the carrageenan-induced leukocyte migration to the peritoneal cavity. Additionally, the carrageenan- and arachidonic acid-induced rat hind paw edema was significantly inhibited (p < 0.05) by i.p. administration of 100 and 200 mg/kg of the compound. When the redox activity was evaluated, CT (200 mg/kg) significantly reduced hepatic lipoperoxidation (p < 0.001), as well as oxidation of plasmatic (p < 0.05) and hepatic (p < 0.01) proteins. The results of the present study support the hypothesis that CT possesses anti-inflammatory and redox protective activities. It is suggested that its effects are associated with the inhibition of the enzymes in the arachidonic acid pathway, which prevent cell migration by inhibiting leukotriene production, edema formation and the increase of reactive oxygen species in tissues. Therefore, CT is of potential benefit to manage inflammatory disorders and correlated damages caused by oxidant agents.