Alterations in sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), and steroid hormone concentrations during pregnancy in rhesus macaques

Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females).     

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.     

Isolation and Characterization of a Virus Inhibitor from Spinach (Spinacia oleracea L.)

A virus inhibiting protein (VI) was isolated from spinach (Spinacia oleracea L.). The VI inhibited infections of test plants with plus- and minus-strand RNA viruses. Inoculation of both local lesion and systemic hosts with TMV in the presence of varying amounts of the VI resulted in typical dose response curves for the number of local lesions or the amount of virus respectively. The lowest concentration of VI leading to a significant reduction in the number of local lesions was 0.06 μg/ml. The VI was found to inhibit local lesion formation only when applied within 2–3 h p.i. but still reduced the number of local lesions when applied up to 9 h prior to virus inoculation. The antiviral activity could be attributed to a protein of molecular weight 29,000 dalton with an isoelectric point of 10.3. Its activity was destroyed by heating for 30 min to 70°C. These characteristics resemble those of other virus inhibiting proteins described for members of the order Caryophyllales such as the Phytolacca inhibitor against which a serological relationship was obtained.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Mode of interference of chlorosis-inducing herbicides with peroxisomal enzyme activities

In rye leaves ( Secale cereale L. cv. Petkus "Kustro") bleached in the presence of the chlorosis-inducing herbicides aminotriazole, haloxidine, San 6706 or difunone in white light of 54.2 W m^-2 (5000 lx), catalase activity was very low. In addition, the activities of glycolate oxidase and hydroxypyruvate reductase were strongly diminished in treatments with San 6706 and difunone. The lowering of the peroxisomal enzyme activities was observed in red, but not in blue light and did not occur after treatment with the non-bleaching pyridazinone derivative San 9785. The deficiencies of the peroxisomal enzymes did not appear to be involved in the initiation of the chlorosis. Instead they are probably produced as secondary consequences of the bleaching. Low peroxisomal enzyme activities were also obtained without herbicide treatment by growing the leaves in an atmosphere of 2% O₂ and 3% CO₂, but in this case were not accompanied by an increased sensitivity of the Chl to photooxidative bleaching. The peroxisomal enzymes reached as high activities as in untreated controls when the herbicide-treated leaves were grown at a low light intensity of 0.106 W m^-2 (10 lx). After transfer of herbicide-treated leaves grown under 0.106 W m^-2 to 306 W m^-2 (30 000 lx), catalase was strongly inactivated, even at 0°C. In treatments with San 6706 and difunone the increase of the activities of glycolate oxidase and hydroxypyruvate reductase was either stopped, remaining unchanged, or the enzymes were slightly inactivated after exposure to 306 W m^-2 (30 000 lx). The observations suggest that the inactivation of peroxisomal enzymes results from photooxidative events in the chloroplasts.     

A role for the thymic epithelium in the selection of pre-T cells from murine bone marrow

A rat thymic epithelial cell line IT45-R1 has been previously described as secreting soluble molecules that in vitro chemoattract rat hemopoietic precursor cells. The development of such an in vitro migration assay was based on the ability of cells to migrate across polycarbonate filters in Boyden chambers. In the present paper, by using the same strategy, we studied murine bone marrow cells capable of migrating in vitro toward IT45-R1 conditioned medium. The responding cells were shown to represent a minor bone marrow subpopulation characterized by a low capacity to incorporate tritiated thymidine in vitro (less than 10% of control). Moreover, this cell subset was considerably impoverished with respect to granulocyte-macrophage CFU (less than 7% of control) and pluripotent hemopoietic stem cells (less than 12% of control). Potential generation of T cells of donor-type in the lymphoid organs of irradiated recipients was measured by using C57BL/Ka Thy-1.1 and Thy-1.2 congenic mice. Thy-1.1 irradiated mice were injected intrathymically or intravenously with the selectively migrated cell subset of Thy-1.2 donor-type bone marrow cells. The use of an i.v. transfer route allowed us to show that these cells possess thymus-homing and colonization abilities. In a time-course study after intrathymic cell transfer, these migrated cells were able to generate Thy-1.2+ donor-type thymocytes represented by all cortical and medullary cell subsets in a single wave of repopulation from day 20 to day 30 after transfer, with a peak around days 23 to 25. The degree of repopulation closely resembled that seen with unfractionated bone marrow cells in terms of absolute numbers of donor cells per thymus (82% of control, 22 x 10(6) Thy-1.2+ cells) as well as in percent donor cells per thymus (105% of control). Thy-1.2+ cells were also detected in the lymph nodes and the spleens of reconstituted recipient mice. Taken together, these results support the idea that the supernatant of the established thymic epithelium IT45-R1 induces the migration of a murine bone marrow subset that contains hemopoietic stem cells already committed to the lymphoid lineage (i.e., pre-T cells).     

Effect of the redox state of QB on electric field-induced charge recombination in Photosystem II

Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts (blebs) by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate Q_B was observed and the earlier failure to detect it is explained. The influence of the Q_B/Q_B ^– oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S₂ and S₃, but not in the state S₄ (actually Z⁺S₃). The presence of the oxidized secondary electron donor, tyrosine Z⁺, appeared to prevent expression of the Q_B/Q_B ^– effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z⁺ and the oxygen evolving complex.Abbreviations BSA bovine serum albumin - EDTA ethylene-diaminetetraacetic acid - EL electroluminescence - FCCP carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone - HEPESI 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - I primary electron acceptor - MOPS 3-(N-morpholino) propane sulfonic acid - P680 primary electron donor of Photosystem II - P700 primary electron donor of Photosystem I - Q_A and Q_B secondary and tertiary electron acceptors of Photosystem II - Z secondary electron donor (D1 Tyr 161)     

Analysis of Fusarium causing dermal toxicosis in marram grass planters

In the European coastal dunes, marram grass (Ammophila arenaria) is planted in order to control sand erosion. In the years 1986 to 1991, workers on the Wadden islands in the Netherlands planting marram grass showed lesions of skin and mucous membranes, suggesting a toxic reaction. Fusarium culmorum dominated the mycoflora of those marram grass culms that were used for planting. This plant material had been cut and stored for more than one week in the open. The Fusarium toxin deoxynivalenol (DON) was detected in the suspect marram grass culms. Isolated F. culmorum strains were able to produce DON in vitro in liquid culture as well as in experimentally inoculated wheat heads. Pathogenicity tests, toxin test as well as RAPD analysis showed that the F. culmorum strains were not specialized for marram grass but may form part of the West-European F. culmorum population infecting cereals and grasses. Storage on old sand-dunes with plant debris may have led to the high occurrence of F. culmorum and contamination with DON. Marram grass culms should be obtained from young plantings on dunes on the seaward slopes and cut culms should not be stored.