Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

Substitution of transferrin by FeCl3 in the development of a low foetal calf serum concentration medium for KB-26.5 hybridoma cell line

KB-26.5, a murine hybridoma cell line producing an IgG₃ monoclonal antibody used in blood type determination, primarily adapted to grow at 5% foetal calf serum (FCS) concentration has been adapted to grow at 0.5% FCS, maintaining its ability to produce antibodies at the same level. In the final step of adaptation, the addition of insulin, transferrin, ethanolamine and selenium to the media formulation was studied, using factorial assay techniques to check the effect of the different compounds and to optimize their required level for satisfactory growth and antibody secretion. KB-26.5 cells required only 20 g/ml of transferrin to adapt to 0.5% FCS medium. Furthermore, transferrin could be substituted by FeCl₃, at a relatively low level of 2 g/ml. Maximum cell density decreased by 31.5% in spinner flask test, but the antibody titer was maintained, thus the specific productivity increased. However, inoculum size had to be increased three-fold with 0.5% FCS medium in order to assure cell growth.     

Kinetics of cooperative ligand–lattice binding: Fast Monte Carlo integration

A fast Monte Carlo integration algorithm with varying time step is described for cooperative binding of ligands of arbitrary length to a one-dimensional lattice. This algorithm is particularly suitable for strongly cooperative or anticooperative systems, i.e., when the time scales for different kinetic events are very different. As an application, the kinetics of a bimodal two-ligand system are briefly discussed.     

Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population.

Two distinct biological phenotypes of human immunodeficiency virus (HIV) have been described: the non-syncytium-inducing (NSI) phenotype, best characterized by the inability to infect MT-2 cells, and the syncytium-inducing (SI) phenotype, with the ability to infect MT-2 cells. The earliest virus population observed following HIV transmission is generally of the NSI phenotype, even after exposure to inocula of mixed NSI/SI phenotype. In this study, the issue of intrapatient selection of virus phenotype following transmission was addressed by studying two cases of accidental transmission. A comparison of the sequences of the V1-V2 and the V3 coding regions of the envelope gene and the p17 region of the gag gene showed that the donor-recipient pairs were tightly clustered in all gene segments, but away from local and published transmission controls. The intrasample variation of the p17 sequence was greater in the recipients and smaller in the donors than that of the V3 region sequence, indicating selection of V3 at transmission. In these transmission cases, the effects of an intravenous inoculation of a small quantity of blood containing predominantly SI V3 sequences (6 of 8 clonal sequences) were compared with those of an intramuscular inoculation of a large quantity of blood containing predominantly NSI viruses (14 of 16 clonal sequences). Both SI and NSI V3 regions were demonstrated to be phenotypic expressions of genetically related viral strains. The inoculation of the predominantly SI virus population resulted in the persistence of an SI virus population in the recipient and a rapid CD4+ T-cell decline. The inoculation of the predominantly NSI population resulted in a selective amplification of SI viruses before seroconversion, followed by a suppression of SI viruses at seroconversion and a rapid decline of CD4+ T-cell numbers. These data suggest that the suppression of SI viruses can be accomplished following the development of HIV-specific immunity and that the ability to suppress SI viruses does not prevent the development of immunodeficiency.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41.

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

Patchiness and disturbance: plant community responses to porcupine diggings in the central Negev

We tested the hypothesis that diversity and productivity of herbaceous plant communities in disturbed soil are related to the physical and biological heterogeneity of the landscape Our study was earned out on vegetation responses in porcupine diggings on a rocky slope in the central Negev desert in Israel We measured aboveground bio-mass and plant density per species in 150 porcupine diggings (15 cm deep and 15 to 20 cm wide) and in equally sized adjacent control samples in the undisturbed soil matrix We calculated mean annual biomass production, plant density and species richness for 10 sample areas along the slope In addition, we divided the plants into groups according to propagule size and dispersal mode We denoted two types of landscape heterogeneity, which we called physical and biological patchiness Physical patchiness was measured as the ratio of bare rock to soil surface Biological patchiness was the area of the soil covered by shrubs with associated soil mound and under-story relative to the total soil surface We also measured disturbance density, as the long term (17 yr) average density of newly made porcupine diggings We found that 1) the physical patchiness explained 30% of the variation of biological patchiness along the slope, while 2) the patterns of disturbance intensity and biological patchiness were similar (R-=0 386) 3) Biomass, density and species richness were significantly higher in diggings than m the soil matrix 4) Plant density in the matrix, but not m the diggings, was significantly correlated with physical patchiness, 5) species richness in diggings was significantly correlated with biological patchiness, but 6) biomass production in diggings and matrix was not affected by either physical or biological patchiness of the landscape 7) Disturbance density did not affect vegetation responses in diggings and matrix 8) A shift in the plant communities in the matrix towards plants with smaller seeds was associated with increasing physical patchiness, while m diggings there was an opposite shift 9) The proportion of wind dispersers was higher in diggings than outside, while the proportion of runoff dispersers was lower, 10) the densities of runoff dispersers in diggings and matrix were positively correlated with physical and biological patchiness 11) Physical and biological patchiness formed the two major gradients of species composition, explaining 30 and 25% respectively We conclude that the network of physical and biological patchiness and soil disturbance are important in the redistribution of resources and seeds, which control plant biomass, density, species richness and diversity The bare rock surface is the main source for runoff flow with associated soil, organic matter and nutrients The understory vegetation of shrubs provides seeds for creating and maintaining diversity The soil matrix absorbs runoff flow, and disturbances absorb runoff and trap seeds Thus, differences in landscape heterogeneity and their effects on resource and seed movement interact in controlling plant community productivity and diversity in the landscape     

Oligosyndactyly: A lethal mutation in the mouse that results in mitotic arrest very early in development

The mutation, oligosyndactyly, results in syndactyly, muscle anomalies, and diabetes insipidus in heterozygous mice. When homozygous, the mutation is lethal early in development. Although homozygous embryos are able to form blastocyst outgrowths (the in vitro equivalent to implantation), cells begin to accumulate in mitosis as early as the blastocyst stage. Even though the cytologic appearance is that of mitotic cells treated with a microtubule inhibitor such as colcemid, the homozygous embryos do, in fact, have normal appearing mitotic spindles. These results define the Os mutation as one which, in the homozygous state, prevents the movement of chromosomes from the metaphase plate. It is the first mammalian developmental mutation to be so defined and is unique among all mitotic arrest mutations thus far described in higher eucaryotes.     

Nd-YAG Laser in Lung Cancer

We administered 45 Nd-YAG laser treatments in 29 patients (18 men) aged 39 to 82 years who had lung malignancy; 26 patients had primary non-oat cell lung cancer and three had metastatic airway malignancy. In all, 25 of the patients had been previously treated with combination(s) of surgical procedure, radiation therapy and chemotherapy. Indications for laser treatment included endobronchial airway obstruction with uncontrolled cough, hemoptysis, dyspnea or unresolved atelectasis-pneumonia. Of 15 patients with partially occluded tracheobronchial airway tumors, immediate palliative relief was achieved in 13 patients and lasted one to six months after a single treatment. In this group there was one postoperative death related to respiratory failure and two patients subsequently died of massive pulmonary hemorrhage. However, of 14 patients with totally obstructed airways, immediate palliative relief was achieved in only five patients and this lasted three weeks to three months after a single treatment. In this group there were two postoperative deaths related to progressive respiratory failure; in one case it was associated with endobronchial combustion of the fiberoptic bronchoscope. All three patients in both groups who died of respiratory failure were in acute respiratory distress and terminally ill before the procedure. These findings suggest that Nd-YAG laser therapy may be most beneficial in patients with partially rather than totally occluded airways due to lung malignancy.     

Mutant HLA-A2 antigens as restricting elements for virus-specific cytotoxic T cells

Mutants of the EB virus-transformed cell line T5-1 (HLA-Al, 2; B8, 27), bearing well-characterized alterations in HLA-A2 antigen expression and unable to bind the HLA-A2-specific monoclonal antibody 13137.2, have been tested for their susceptibility to EB virus-specific cytolysis using effector T-cell preparations functionally restricted through relevant HLA antigens. Initial experiments first confirmed that the parent line T5-1 was susceptible to cytolysis by both common A2-restricted and 1327-restricted effector cells. While those T5-1 mutants with little or no surface A2 expression were not lysed by A2-restricted effectors, those targets with quantitatively normal expression of mutant A2 molecules were as susceptible to A2-restricted lysis as the parent line itself. In contrast, all the T5-1 mutant lines were susceptible to B27-restricted cytolysis. The results demonstrate that experimentally induced mutations of HLA-A2 antigen structure, affecting a serologically defined site on the molecule, can occur without altering that same molecule's expression of the T cell-restricting determinant(s). Such experimentally induced mutations are quite different from the naturally occurring variant A2 antigens which are present within the serologically defined A2 antigen group and which show changes at the T cell-restricting site.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.