Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Life history adaptations of Gasterosteus aculeatus in a Mediterranean wetland

Synopsis The life cycle of leiurus-type Gasterosteus aculeatus occurring in a Mediterranean coastal wetland is described. Fish have a low number of lateral plates, short spines and marked sexual dimorphism in size. The life cycle is strictly annual, adults dying shortly after breeding. Adult fish migrate into seasonally-flooded freshwater marshes to breed, and the young migrate back to brackish water to pass the summer and autumn. Breeding occurs in March at water temperatures of about 10°C, the season lasting about 50 days. Growth of fish occurs throughout the year, but differs from year to year, resulting in variable adult size. Maximum gonadal investment of male fish is in autumn, whereas that of females is in spring. Gonadal investment of female fish, as measured by gonado-somatic index and fecundity, is higher than in other studied leiurus populations, but the number of clutches produced in a season is probably low. These differences in life history from other studied populations of sticklebacks are seen as adaptations to a mediterranean-type climate (high summer temperatures, seasonality of water bodies) and to heavy predation by fish-eating birds.     

Luminal hydrolysis of recombinant human epidermal growth factor in the rat gastrointestinal tract: segmental and developmental differences

Epidermal growth factor (EGF), present in high concentrations in the milk of various species, is biologically active following oral administration to young animals. Although in vivo studies show gastrointestinal processing of dietary EGF during early postnatal development, the relative importance of luminal and mucosal digestion in such processing is undefined. To characterize the luminal metabolism of dietary EGF in the developing gastrointestinal tract, we incubated human recombinant 125I-EGF in vitro at 37 degrees with luminal fluid from the stomach and various segments of the small intestine of 12 day old suckling and 31 day old weanling rats and analyzed the resulting reaction products. The rate of EGF hydrolysis as determined by generation of acid soluble material was greater in weanling small intestine than in suckling, with maximal hydrolytic capacity observed in the mid-jejunum and ileum. Minimal hydrolysis was observed with stomach fluid from both age groups, and EGF retained its ability to elute as a single species on Sephadex G-25 columns and to bind to monospecific affinity columns and placental membrane receptors. Incubation with suckling small intestinal fluid produced little change in the chromatographic profile on Sephadex G-25, but a reduction in antibody and receptor binding was observed. In contrast, incubation with weanling small intestinal fluid yielded both a more pronounced loss of EGF-like material on G-25 columns and a greater reduction in receptor and antibody binding. We conclude that little luminal EGF degradation occurs in the rat stomach during the suckling and weanling periods, but that in the lumen of the small intestine breakdown increases during postnatal development.     

Activating and 'anxiogenic' effects of corticotropin releasing factor are not inhibited by blockade of the pituitary-adrenal system with dexamethasone

Central administration of corticotropin releasing factor (CRF) in rats produces pituitary-adrenal activation and a variety of "anxiogenic-like" effects. The present study was designed to explore the contribution of the peripheral pituitary-adrenocortical axis in mediating these CRF responses. Intraventricularly administered CRF produced suppression of responding in the conflict test and a marked locomotor activation. Neither behavioral effect was altered by the prior administration of dexamethasone in a dose that blocked pituitary-adrenal activation to CRF. These results support the hypothesis that behavioral effects of CRF are mediated by its action at central sites and not via an action on the pituitary-adrenocortical system.     

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: characterization of the cleavage by solubilized endopeptidase-24.11

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly29-Trp30, Trp30-Met31 and Asp32-Phe33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.     

Distribution of carotenoids in sub-cellular and sub-plastidic fractions of radish seedlings (Raphanus sativus) grown in the presence of bleaching herbicides

The intracellular and intraplastidic distribution of carotenoids has been investigated in radish seedlings grown in the presence of the herbicides amitrole and SAN 6706. Both herbicides caused bleaching and the plants became deficient in chlorophylls and the usual chloroplast cyclic carotenoids, but accumulated the acyclic carotenoid biosynthetic intermediates 15-cis-phytoene and all-trans-lycopene. In both the untreated and herbicide-treated plants all carotenoids, including phytoene and lycopene, were contained in the plastid. In all cases the normal cyclic carotenoids were located virtually exclusively in the thylakoid or prothylakoid fraction. In amitrole-treated plants, lycopene also was contained only in the thylakoid fraction, whereas phytoene, in these and in SAN 6706-treated plants, was detected in both the thylakoid fraction and an envelope preparation. Possible implications for the biosynthesis of the carotenoids are discussed.     

Similarities in the heavy and light chains of tetanus toxin suggested by their amino acid compositions

Quantitative comparison of the amino acid compositions of the heavy and light chains of tetanus toxin by the method of Cornish-Bowden [(1983) Methods Enzymol. 91, 60-75)] suggests strongly that there is sequence homology between the two chains and that the heavy chain has two similar halves. Examination (by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate) of peptides produced from the chains by proteolytic cleavage supports this idea.     

Dipstick haematuria and bladder cancer in men over 60: results of a community study.

OBJECTIVE--To investigate the prevalence and relevance of dipstick haematuria in a group of men in the community. DESIGN--Prospective study of elderly men invited to attend a health centre for urine screening as part of a health check. SETTING--An inner city health centre in Leeds. SUBJECTS--578 Of 855 men aged 60-85 responding to an invitation to participate. INTERVENTIONS--The subjects had their urine tested with a dipstick (Multistix) for the presence of blood and then tested their urine once a week for the next 10 weeks. Those with one or more positive test results were offered full urological investigation. MAIN OUTCOME MEASURE--The prevalence of urological disease in those subjects with dipstick haematuria. RESULTS--78 Men (13%) had dipstick haematuria on a single test and a further 54 (9%) had evidence of dipstick haematuria when testing their urine once a week during a subsequent 10 week period. Investigation of 87 men disclosed urological disease in 45, including four with a bladder tumour and seven with epithelial dysplasia. CONCLUSION--Dipstick haematuria is a common incidental finding in men over 60 and is associated with appreciable urological disease. The introduction of less invasive methods of investigation, particularly flexible cystoscopy and ultrasonography, has made investigation of these patients simple and safe and makes screening for bladder cancer in the community more feasible.     

Photobleaching in the unicellular green alga Dunaliella parva 19/9

The change in the pigment composition of the unicellular alga Dunaliella parva 19/9 during exposure to high light (4000 mol m^-2 s^-1) has been investigated. During photobleaching the carotenoids were lost at a greater rate than the chlorophylls. In these photoinhibitory conditions, -carotene and especially the minor carotenes, - and -carotene, were more susceptible to oxidative destriction than the xanthophylls. Lutein, the major carotenoid present, was the most stable of the carotenoids in these conditions. In addition to the direct photobleaching of carotenoids and chlorophylls, high light treatment induced the de-epoxidation of violaxanthin to antheraxantin and zeaxanthin. Small amounts of zeaxanthin were present in cells prior to illumination but the amount increased 2.4 fold following high light treatment. The effects of extremes of temperature during exposure to high light intensities were also investigated. The destruction of chlorophylls was found to be more temperature sensitive than that of the carotenoids. The general pattern of loss for the individual carotenoids was similar to that found at 25°C, i.e., the carotenes were more readily degraded than the xanthophylls.