Xylitol production by immobilized recombinant Saccharomyces cerevisiae in a continuous packed-bed bioreactor

Continuous xylitol production with two different immobilized recombinant Saccharomyces cerevisiae strains (H475 and S641), expressing low and high xylose reductase (XR) activities, was investigated in a lab-scale packed-bed bioreactor. The effect of hydraulic residence time (HRT; 1.3-11.3 h), substrate/cosubstrate ratio (0.5 and 1), recycling ratio (0, 5, and 10), and aeration (anaerobic and oxygen limited conditions) were studied. The cells were immobilized by gel entrapment using Ca-alginate as support and the beads were treated with Al(3+) to improve their mechanical strength. Xylose was converted to xylitol using glucose as cosubstrate for regeneration of NAD(P)H required in xylitol formation and for generation of maintenance energy. The stability of the recombinant strains after 15 days of continuous operation was evaluated by XR activity and plasmid retention analyses. Under anaerobic conditions the volumetric xylitol productivity increased with decreasing HRT with both strains. With a recycling ratio of 10, volumetric productivities as high as 3.44 and 5.80 g/L . h were obtained with the low XR strain at HRT 1.3 h and with the high XR strain at HRT 2.6 h, respectively. However, the highest overall xylitol yields on xylose and on cosubstrate were reached at higher HRTs. Lowering the xylose/cosubstrate ratio from 1 to 0.5 increased the overall yield of xylitol on xylose, but the productivity and the xylitol yield on cosubstrate decreased. Under oxygen limited conditions the effect of the recycling ratio on production parameters was masked by other factors, such as an accumulation of free cells in the bioreactor and severe genetic instability of the high XR strain. Under anaerobic conditions the instability was less severe, causing a decrease in XR activity from 0.15 to 0.10 and from 3.18 to 1.49 U/mg with the low and high XR strains, respectively. At the end of the fermentation, the fraction of plasmid bearing cells in the beads was close to 100% for the low XR strain; however, it was significantly lower for the high XR strain, particularly for cells from the interior of the beads. (c) 1996 John Wiley & Sons, Inc.     

Inheritance of random amplified polymorphic DNA markers in cassava (Manihot esculenta Crantz)

The informativeness and inheritance of randomly amplified polymorphic DNA (RAPD) markers were investigated in an intraspecific F1 progeny derived from two heterozygous parents. The analysis confirmed the utility of RAPD markers for comparing candidate parents for the development of a molecular genetic map, and provided numerous markers for linkage analysis in a crop with a very limited history of classical or molecular genetic studies. Six potential parental lines (themselves F1 hybrid clones) showed between 1.82 and 0.62 segregating bands per primer in three hybrid families. Forty-three percent (309) of 722 primers produced polymorphic products in the most informative of these three crosses, revealing 328 single-dose (SD) markers segregating 1:1 for presence/absence in a progeny of 90 individuals. A second class of informative markers were those present in both parents but segregating in the progeny. Fifty-seven or 67% of the monomorphic but segregating markers exhibited the 3:1 ratio expected for SD dominant markers in a cross between heterozygotes. Linkage groups were constructed from the segregation of SD RAPD markers originating in the female (TMS 30572) and the male (CM2177-2) parent. Key words : RAPDs, molecular markers, genetic segregation, Manihot, single-dose markers.     

A New Type of Manchette Structure in Spermatids of Culex tigripes(Culicidae)

Abstract In young spermatids of Culex tigripes, microtubules organize a manchette which surrounds the nucleus. When the nucleus elongates, 1–5 expansions appear on the wall of the microtubules. They grow and branch out while the nucleus elongates and chromatin condenses. Expansions are straight or curved in shape. They have the same thickness as the microtubule walls, but they rarely constitute links between microtubules. The manchette disappears naturally at the end of spermiogenesis. The action of colchicine on spermatids leads to the complete disappearance of the microtubules and expansions, and inhibits the lengthening of the nucleus.     

Starvation-Induced Stress Resistance in Lactococcus lactis subsp. lactis IL1403

Carbohydrate-starved cultures of Lactococcus lactis subsp. lactis IL1403 showed enhanced resistance to heat, ethanol, acid, osmotic, and oxidative stresses. This cross-protection seems to be established progressively during the transitional growth phase, with maximum resistance occurring when cells enter the stationary phase. Chloramphenicol or rifamycin treatment does not abolish the development of a tolerant cell state but, on the contrary, seems to provoke this response in L. lactis subsp. lactis.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.     

Sperm concentration influences fertilization and male pronuclear formation in vitro in pigs

To study the effect of sperm concentration on the results of pig in vitro fertilization (IVF), 313 oocytes recovered from oviducts of prepubertal gilts after induction of ovulation were used. After capacitation, the number of live spermatozoa in the fertilization dishes was adjusted to 3 x 10(5), 6 x 10(5) and 12 x 10(5) cell/ml. After 4 hours of co-culture in TCM-199, the oocytes were pippeted to remove cumulus cells and the excess spermatozoa around the zona pellucida, and were transferred to fresh TCM-199 for another 12 14 hours . The results showed that 6 x 10(5) spermatozoa/ml is the optimum concentration for this system; the percentage of fertilized ova (71.6%) was not different from the best (76.8%), that was obtained with the highest concentration, and the percentage of monospermy (62.3%) was not different from the best (68.1%), that was obtained with the lowest concentration. The percentage of spermatozoa that reached the pronuclear stage increased while sperm concentration was decreased. The percentage of spermatozoa at the decondensed stage was decreased when the sperm concentration increased.     

Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

Ecosystem-level effects of re-oligotrophication and N:P imbalances in rivers and estuaries on a global scale

Trends and ecological consequences of phosphorus (P) decline and increasing nitrogen (N) to phosphorus (N:P) ratios in rivers and estuaries are reviewed and discussed. Results suggest that re-oligotrophication is a dominant trend in rivers and estuaries of high-income countries in the last two–three decades, while in low-income countries widespread eutrophication occurs. The decline in P is well documented in hundreds of rivers of United States and the European Union, but the biotic response of rivers and estuaries besides phytoplankton decline such as trends in phytoplankton composition, changes in primary production, ecosystem shifts, cascading effects, changes in ecosystem metabolism, etc., have not been sufficiently monitored and investigated, neither the effects of N:P imbalance. N:P imbalance has significant ecological effects that need to be further investigated. There is a growing number of cases in which phytoplankton biomass have been shown to decrease due to re-oligotrophication, but the potential regime shift from phytoplankton to macrophyte dominance described in shallow lakes has been documented only in a few rivers and estuaries yet. The main reasons why regime shifts are rarely described in rivers and estuaries are, from one hand the scarcity of data on macrophyte cover trends, and from the other hand physical factors such as peak flows or high turbidity that could prevent a general spread of submerged macrophytes as observed in shallow lakes. Moreover, re-oligotrophication effects on rivers may be different compared to lakes (e.g., lower dominance of macrophytes) or estuaries (e.g., limitation of primary production by N instead of P) or may be dependent on river/estuary type. We conclude that river and estuary re-oligotrophication effects are complex, diverse and still little known, and in some cases are equivalent to those described in shallow lakes, but the regime shift is more likely to occur in mid to high-order rivers and shallow estuaries.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

STADEERS: a software package for the statistical design of experiments pertaining to the estimation of parameters in rate expressions that describe enzyme-catalyzed processes

This paper describes a computational algorithm (STADEERS-STAtisticalDesign of Exeriments in Enzyme ReactorS) for the statisticaldesign of biochemical engineering experiments. The type of experimentthat qualifies for this package involves a batch reaction catalyzedby a soluble enzyme where the activity of the enzyme decayswith time. Assuming that both the catalytic action and the deactivationof the enzyme obey known rate expressions, the present codeis helpful in the process of obtaining estimates of the kineticparameters by providing as output the times at which samplesshould be withdrawn from the reacting mixture. Starting D-optimaldesign is used as a basis for the statistical approach. ThisBASIC code is a powerful tool when fitting a rate expressionto data because it increases the effectiveness of experimentationby helping the biochemical kineticist obtain data points withthe largest possible informa tional content.