Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Purification and Characterization of Soluble (Cytosolic) and Bound (Cell Wall) Isoforms of Invertases in Barley (Hordeum vulgare) Elongating Stem Tissue

Three different isoforms of invertases have been detected in the developing internodes of barley (Hordeum vulgare). Based on substrate specificities, the isoforms have been identified to be invertases (β-fructosidases EC 3.2.1.26). The soluble (cytosolic) invertase isoform can be purified to apparent homogeneity by diethylaminoethyl cellulose, Concanavalin-A Sepharose, organomercurial Sepharose, and Sephacryl S-300 chromatography. A bound (cell wall) invertase isoform can be released by 1 molar salt and purified further by the same procedures as above except omitting the organo-mercurial Sepharose affinity chromatography step. A third isoform of invertase, which is apparently tightly associated with the cell wall, cannot be isolated yet. The soluble and bound invertase isoforms were purified by factors of 60- and 7-fold, respectively. The native enzymes have an apparent molecular weight of 120 kilodaltons as estimated by gel filtration. They have been identified to be dimers under denaturing and nondenaturing conditions. The soluble enzyme has a pH optimum of 5.5, K_m of 12 millimolar, and a V_max of 80 micromole per minute per milligram of protein compared with cell wall isozyme which has a pH optimum of 4.5, K_m of millimolar, and a V_max of 9 micromole per minute per milligram of protein.     

Localization and Pattern of Graviresponse across the Pulvinus of Barley Hordeum vulgare

Pulvini of excised stem segments from barley (Hordeum vulgare cv `Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0° and the lowermost point 180°. A gravity-induced increase in pulvinus thickness was observable within 40° of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.     

Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods

Of the biogeochemical processes, denitrification has perhaps been the most difficult to study in the field because of the inability to measure the product of the process. The last decade of research, however, has provided both acetylene and¹⁵N based methods as well as undisturbed soil core andin situ soil cover sampling approaches to implementing these methods. All of these methods, if used appropriately, give comparable results. Thus, we now have several methods, each with advantages for particular sites or objectives, that accurately measure denitrification in nature. Because of the general usefulness of the acetylene methods, updated protocols for the following three methods are given: gas-phase recirculation soil cores; static soil cores; and the denitrifying enzyme assay also known as the phase 1 assay. Despite the availability of these and other methods, denitrification budgets remain difficult to accurately establish in most environments because of the high spatial and temporal variability inherent in denitrification. Appropriate analysis of those data includes a distribution analysis of the data, and if highly skewed as is typically the case, the most accurate method to estimate the mean and the population variance is the UMVUE method (uniformly minimum variance unbiased estimator). Geostatistical methods have also been employed to improve spatial and temporal estimates of denitrification. These have occasionally been successful for spatial analysis but in the attempt described here for temporal analysis the approach was not useful.Discussions of the importance of denitrification have always focused on quantifying the process and whether particular measured quantities are judged to be a significant amount of nitrogen. A second line of evidence discussed here is the extant genetic record that results from natural selection. These analysis lead to the conclusion that strong selection for denitrification must currently be occurring, which implies that the process is of general significance in soils.     

A ten-month study of endogenous testosterone levels and behaviour in outdoor-living female rhesus monkeys (Macaca mulatta)

Endogenous testosterone levels were measured in association with sexual, aggressive, and social/affiliative behaviors in 11 outdoor-housed female rhesus monkeys over a ten-month period. Several behaviors (sex directed toward the male, sex received from the male, aggression directed toward the male, submission directed toward the male, submission directed toward the female, and groom another female) were significantly (p<0.05) positively correlated with testosterone in from one to five females. No trends were strong enough across all females to suggest that any of these correlations have species-wide significance. Factor analysis revealed clearcut clusters of behaviors, but elevations in testosterone were not strongly associated with any of these clusters. It is concluded that endogenous testosterone levels have little measurable effect on overt behavior in female rhesus monkeys.     

Cytoskeletal involvement in spermiation and sperm transport

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.     

Mucosal epithelial cells and Langerhans cells are targets for infection by the immunosuppressive type D retrovirus simian AIDS retrovirus serotype 1

Simian acquired immune deficiency syndrome (SAIDS) caused by the type D retrovirus SRV-1 results in opportunistic infections and a spectrum of oral lesions similar to those seen in humans with AIDS. To better understand the pathogenesis of these oral lesions we have retrospectively examined the oral mucosa from ten rhesus monkeys that died with SAIDS and prospectively examined the oral mucosa of ten additional animals inoculated with SRV-1 to determine at what time, and in what cells SRV-1 infection of the oral mucosa occurs. Using single and double label immunohistologic techniques, and electron microscopy we detected SRV-1 in clusters of oral epithelial cells and rare Langerhans cells as early as 1 month postinoculation.     

Computational experience with an algorithm for tetrangle inequality bound smoothing

An important component of computer programs for determining the solution conformation of proteins and other flexible molecules from nuclear magnetic resonance data are the so-called “bound smoothing algorithms”, which compute lower and upper limits on the values of all the interatomic distances from the relatively sparse set which can usually be measured experimentally. To date, the only methods efficient enough for use in large problems take account of only the triangle inequality, but an appreciable improvement in the precision of the limits is possible if the algebraic relations between the distances among each quadruple of atoms are also considered. The goal of this paper is to use a recently improved algorithm for computing these “tetrangle inequality limits” to determine just how much improvement really is possible, given the types of experimental data that are usually available.     

Temporal changes in serum prostaglandin F2alpha and oxytocin in dairy cows with short luteal phases after the first postpartum ovulation

Luteolysis in the cow depends upon an interaction between prostaglandin F(2alpha) (PGF(2alpha)) and oxytocin. The objectives of our study were 1) to determine oxytocin concentrations in postpartum dairy cows and 2) to identify the temporal relationship between oxytocin and PGF(2alpha) release patterns during luteolysis in normal and abbreviated estrous cycles in the postpartum period. Serum oxytocin and PGF(2alpha) metabolite (PGFM) concentrations from nine cows which had short estrous cycles (< 17 d) were compared with those of six cows which had normal estrous cycles. Serum basal oxytocin concentrations in short estrous cycle cows (23.7 to 31.1 pg/ml) were higher (P<0.05) than those of normal estrous cycle cows (14.6 to 19.8 pg/ml). Oxytocin concentrations increased to peak values in both short and normal cycle cows, during luteolysis. Basal PGFM concentrations (112.2 to 137.4 pg/ml) were higher in cows with short cycle (P<0.05) than in cows with normal cycles (62.9 to 87.5 pg/ml). The increase in PGFM concentrations during luteolysis was significant in both normal cycle and short cycle cows (P<0.05). Increases in serum PGFM concentrations were always associated with increases in serum oxytocin concentrations in normal cycle and short cycle cows and the levels decreased simultaneously before the subsequent estrus. Results support the idea of a positive relationship between PGF(2alpha) and oxytocin concentration during the estrous cycle as well as a possible synergistic action of these hormones in the induction of luteolysis in dairy cattle.