A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Cation-independent Mannose 6-Phosphate Receptor: A COMPOSITE OF DISTINCT PHOSPHOMANNOSYL BINDING SITES*

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting ∼60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5–9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1–3, interacts with Man₈GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes.     

Extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-T-lymphocyte response

Selection for escape mutant immunodeficiency viruses by cytotoxic T lymphocytes (CTL) has been well characterized and may be associated with disease progression. CTL epitopes accrue escape mutations at different rates in vivo. Interestingly, certain high-frequency CTL do not select for escape until the chronic phase of infection. Here we show that mutations conferring escape from immunodominant CTL directed against an epitope in the viral Gag protein are strongly associated with extraepitopic mutations in gag in vivo. The extraepitopic mutations partially restore in vitro replicative fitness of viruses bearing the escape mutations. Constraints on epitope sequences may therefore play a role in determining the rate of escape from CTL responses in vivo.     

Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKCdelta

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-1 (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased Ca(2+) sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-IH augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent Ca(2+) sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 microM) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 microM) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor G?-6976 (1 microM) had no effect on ET-1-mediated vasoconstriction in either group, but a PKCdelta-selective inhibitor (rottlerin, 3 microM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKCdelta phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by G?-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKCdelta activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.     

A biochemical basis for induction of retina regeneration by antioxidants

The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. Embryonic chicks are able to regenerate their retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye only if a source of exogenous factors, such as FGF2, is present. This study shows that NAC modifies the redox status of the CM, initiates self-renewal of the stem/progenitor cells, and induces regeneration in the absence of FGF2. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with different biochemical structures and modes of action, and found that NAC induces regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway.     

Combined effects of night-time temperature and allelochemicals on performance of a generalist insect herbivore

To assess the pattern of temperature influencing the effect of allelochemicals on growth of insect herbivores and to examine the potential effect of warmer nights due to global warming, we examined the simultaneous effects of allelochemicals and warmer night-time temperatures on an insect herbivore (Spodoptera exigua; Lepidoptera: Noctuidae). Dietary chlorogenic acid, rutin and tomatine levels reflected those occurring naturally in the leaves of tomato, a hostplant of this herbivore. We compared the effects of four thermal regimes having a daytime temperature of 26 °C , with the night-time temperature increased from 14 to 26 °C by increments of 4 °C . The effect of a particular allelochemical on developmental rate was similar among the four thermal regimes. Chlorogenic acid and tomatine each reduced final larval weight, but there was no effect of night-time temperature. In contrast, rutin had no effect on final weight, whereas final weight declined with increasing night-time temperature. Night-time temperature did not influence amount eaten. Larvae ate less when chlorogenic acid or tomatine was in the diet. For each allelochemical, there were no allelochemical by thermal regime interactions. In addition, we compared the effects of allelochemicals and the thermal regime of 26:14 °C and constant 20 °C , which was the average temperature of the 26:14 °C regime. Developmental rate was lower at the constant 20 °C regime, chlorogenic acid and tomatine each depressed developmental rate, and there were no allelochemical by thermal regime interactions. Thus, regardless of the specific allelochemical or amount, the pattern of response at the fluctuating regime was similar to that at the constant temperature. In contrast, comparison of the thermal regime of 26:22 °C and constant 24 °C , which was the average temperature of the 26:22 °C regime, showed several allelochemical by thermal regime interactions. At the 26:22 °C regime, developmental rate was disproportionatly higher at the maximal rutin concentration compared to that at constant 24 °C . At the constant 24 °C , final larval mass was disproportionately lower at the moderate tomatine concentration compared to that at the 26:22 °C regime. Because these results differ from that of other studies examining another species, it appears that the response to incremental changes in night-time temperature will reflect the allelochemicals and insect species tested. The contrast between the constant 24 °C and 26:22 °C regimes indicates that even small fluctuations (±2 °C ) in temperature over 24 h can yield differences in the response to an allelochemical.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

Material Spiraling in Stream Corridors: A Telescoping Ecosystem Model

Stream ecosystems consist of several subsystems that are spatially distributed concentrically, analogous to the elements of a simple telescope. Subsystems include the central surface stream, vertically and laterally arrayed saturated sediments (hyporheic and parafluvial zones), and the most distal element, the riparian zone. These zones are hydrologically connected; thus water and its dissolved and suspended load move through all of these subsystems as it flows downstream. In any given subsystem, chemical transformations result in a change in the quantity of materials in transport. Processing length is the length of subsystem required to “process” an amount of substrate equal to advective input. Long processing lengths reflect low rates of material cycling. Processing length provides the length dimension of each cylindrical element of the telescope and is specific to subsystem (for example, the surface stream), substrate (for instance, nitrate), and process (denitrification, for example). Disturbance causes processing length to increase. Processing length decreases during succession following disturbance. The whole stream-corridor ecosystem consists of several nested cylindrical elements that extend and retract, much as would a telescope, in response to disturbance regime. This telescoping ecosystem model (TEM) can improve understanding of material retention in running water systems; that is, their “nutrient filtration” capacity. We hypothesize that disturbance by flooding alters this capacity in proportion to both intensity of disturbance and to the relative effect of disturbance on each subsystem. We would expect more distal subsystems (for example, the riparian zone) to show the highest resistance to floods. In contrast, we predict that postflood recovery of functions such as material processing (that is, resilience) will be highest in central elements and decrease laterally. Resistance and resilience of subsystems are thus both inversely correlated and spatially separated. We further hypothesize that cross-linkages between adjacent subsystems will enhance resilience of the system as a whole. Whole-ecosystem retention, transformation, and transport are thus viewed as a function of subsystem extent, lateral and vertical linkage, and disturbance regime. Received 15 April 1997; accepted 1 September 1997.     

Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase

The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.