The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

Negative surface charge density near heart calcium channels. Relevance to block by dihydropyridines

We have measured the density of negative surface charges near the voltage sensor for inactivation gating of (L-type) Ca channels in intact calf Purkinje fibers and in isolated myocytes from guinea pig and rat ventricles. Divalent cation-induced changes in the half-maximal voltage for inactivation were determined and were well described by curves predicted by surface potential theory. We measured shifts in inactivation induced by Ca, Sr, and Ba in the single cells, and by Sr in the Purkinje fibers. All of the data were consistent with an estimated negative surface charge density of 1 electronic charge per 250 A2. In addition, the data suggest that Ca, but neither Ba nor Sr, binds to the negative charges with an association constant on the order of 1 M-1. We find that divalent ion-induced changes in surface potential can account for most of the antagonism between these ions and Ca channel block by 1,4-dihydropyridines.     

Isolation and characterization of the two major apoproteins in human lipoprotein [a]

Human Lp[a] was isolated in preparative amounts from two donors; the native lipoprotein and its constituent apoproteins, apo[a] and apoB, were characterized extensively. Based on differences in apparent molecular weight, four different isoforms of apo[a], a1-a4, were observed between the two donors. The number and relative distribution of these isoforms varied between donors but were constant for each donor. Each apo[a] isoform was shown to be derived from a discrete apo[a]-B100 disulfide-linked complex present before reduction. Complete delipidation of Lp[a] was followed by solubilization, reduction, and carboxamidomethylation of the constituent apoproteins. These apoproteins were then separated by immunoaffinity chromatography using anti-apo[a]- or anti-apoB-Sepharose; their purity and structural integrity were demonstrated by Western blot analysis. ApoB isolated by this procedure was essentially identical to apoB from autologous LDL with respect to molecular weight, secondary structure, amino acid composition, and sialic acid content. However, apo[a] differed from apoB in that it exhibited: a much less alpha-helical, less beta, but much more disordered structure; a lower proportion of aspartate, isoleucine, leucine, phenylalanine, and lysine, but a higher proportion of proline, glycine, and threonine; and a much higher content of sialic acid. These results indicate that apo[a] is not a superglycosylated form of apoB but is distinctly different in its composition and structure.     

Quantitative assessment of vaginal microflora during use of tampons of various compositions.

Although the effect of vaginal tampons on the microbial flora during menstruation has recently been studied by several investigators, quantitative effects attributable to particular tampon fibers have received less attention. The purposes of the present study were (i) to determine and then to compare the effects of polyacrylate rayon tampons and viscose rayon tampons on the normal vaginal flora, (ii) to compare quantitative bacterial counts obtained from these tampons with those obtained from concomitant vaginal swabs, and (iii) to determine whether either of these tampon types alters the vaginal microflora when compared with the microflora in the same women using all-cotton tampons or external catamenial pads. Tampon and swab samples were obtained at predetermined times from 18 women for an average of seven menstrual cycles. Samples consisting of swabs from women wearing menstrual pads were compared with swab and tampon samples taken at predetermined times during the menstrual cycle from women using cotton, polyacrylate rayon, or viscose rayon tampons. Samples were analyzed for total aerobic, facultative, and anaerobic bacterial counts. Statistical evaluation of the results indicated that, on the whole, total bacterial counts decreased during menstruation and that the numbers of bacteria in tampons tended to be lower than those in swab samples taken at the same time. The tampon type had little effect on the vaginal microflora.     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

The eccentric cell of theLimulus lateral eye: encoder of circadian changes in visual responses

Efferent fibers from a central circadian clock innervate both photoreceptor cells and second-order neurons (eccentric cells) in the lateral compound eye ofLimulus, and release octopamine when activated. We have used intracellular microelectrodes to study the modulation of eccentric cell function by efferent optic-nerve activity, octopamine agonists, and a K⁺-channel blocker, TEA.

Identification of lymphocyte subpopulations with a polymethine dye

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.     

Phosphorus availability under alley cropping and mulched and unmulched sole cropping systems in Costa Rica

Phosphorus availability was measured in soils under five cropping systems: alley cropping with Erythrina poeppigiana, alley cropping with Gliricidia sepium, sole cropping with Erythrina poeppigiana mulch applied, sole cropping with Gliricidia sepium mulch applied, sole cropping with no mulch. The following parameters were measured: 1) plant-available soil P assessed by P uptake of maize and bean bioassay plants; 2) phosphate desorbable by anion exchange resin; 3) adsorption of added P into isotopically exchangeable and non-exchangeable pools.In the bioassay, P uptake of beans declined in the order: mulched sole-cropped>unmulched sole-cropped>alley-cropped soils. For maize the relative uptake was: mulched sole-cropped>unmulched sole-cropped = alley-cropped soils. These results suggest trees had not incorporated a significant quantity of P into the system after seven years and, probably, there was a decrease in available soil P due to the sequestration of P in the tree biomass. Potentially resin-desorbable P was higher in alley-cropped and mulched sole-cropped soils than in unmulched sole-cropped soils. The adsorption and desorption of added P into and from exchangeable and non-exchangeable pools did not differ between alley-cropped and unmulched sole-cropped soils.Crop yield and crop N, P and K uptake were all higher in the alley crops than in the unmulched sole crop. The supply of P to the crop under alley cropping seems to be dependent on P cycled and released from the mulch. The P cycle in alley cropping appears to be self-sustaining at least under conditions of moderate P fertiliser input.