Conditional lethality yields a new vaccine strain of Listeria monocytogenes for the induction of cell-mediated immunity

Listeria monocytogenes is a gram-positive intracellular pathogen that can enter phagocytic and nonphagocytic cells and colonize their cytosols. Taking advantage of this property to generate an intracellular vaccine delivery vector, we previously described a mutant strain of L. monocytogenes, Deltadal Deltadat, which is unable to synthesize cell wall by virtue of deletions in two genes (dal and dat) required for d-alanine synthesis. This highly attenuated strain induced long-lived protective systemic and mucosal immune responses in mice when administered in the transient presence of d-alanine. We have now increased the usefulness of this organism as a vaccine vector by use of an inducible complementation system that obviates the need for exogenous d-alanine administration. The strain expresses a copy of the Bacillus subtilis racemase gene under the control of a tightly regulated isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible promoter present on a multicopy plasmid. This bacterium demonstrates strict dose-dependent growth in the presence of IPTG. After removal of inducer, bacterial growth ceased within two replication cycles. Following infection of mice in the absence of IPTG or d-alanine, the bacterium survived in vivo for less than 3 days. Nevertheless, a single immunization elicited a state of long-lasting protective immunity against wild-type L. monocytogenes and induced a subset of effector listeriolysin O-specific CD11a(+) CD8(+) T cells in spleen and other tissues that was strongly enhanced after secondary immunization. This improved L. monocytogenes vector system may have potential use as a live vaccine against human immunodeficiency virus, other infectious diseases, and cancer.     

Perceived exertion and blood lactate concentration during graded treadmill running

The purpose of this study was to investigate the influences of treadmill gradients on the rating of perceived exertion (RPE) at two fixed blood lactate concentrations ( [La^–]_b). Ten subjects performed three different incremental treadmill protocols by running either uphill (concentrically-biased), downhill (eccentrically-biased), or on the flat (non-biased). Individual data of each protocol were interpolated to reflect [La^–]_b corresponding to 2.0 and 4.0 mmol·l^–1. At 2.0 mmol·l^–1 [La^– _b, RPE and treadmill speed during downhill running were greater than during level running which was greater than during uphill running (p < 0.05) . Also, the downhill heart rate (HR) was greater than the uphill HR, and downhill minute ventilation ( ) was greater than the level . Treadmill speed was the only measure at 4.0 mmol·l^–1 [La^–]_b to differ between gradients. There was a moderate correlation of RPE with HR at both [La^–]_b (r = 0.73 at 2.0 mmol·l^–1;r = 0.48 at 4.0 mmol·l^–1) while treadmill speed was moderately correlated with RPE only at 2.0 mmol·l^–1 [La^–]_b (r = 0.70). The results of this study demonstrated that the degree of eccentric-bias during running exercise is an influence of perceived exertion at a moderate but not at a high exercise intensity.     

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP ⁺ transformants). Plasmids isolated from the transformants (URA ⁺ TRP ⁺) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.     

Chimeric Clostridial Cytotoxins: Identification of the N-Terminal Region Involved in Protein Substrate Recognition

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.     

Evaluation of the Chemical Composition of Dacryodes Edulis and Raphia Hookeri Mann and Wendl Exudates used in Herbal Medicine in South Eastern Nigeria

The phytochemical contents and medicinal values of Dacroydes edulis and Raphia hookeri exudates were investigated. Phytochemical screening of the plant showed that they contain the presence of bioactive compounds comprising saponins (2.08–3.98mg 100g⁻¹), alkaloids (0.28–0.49 mg 100g⁻¹), tannins (0.47–0.72 mg 100g⁻¹), flavonoids (0.26–0.39 mg 100g⁻¹), and phenolic compounds (0.01–0.05 mg 100g⁻¹). The carbohydrates, lipids and protein content were 77.42–78.90%, 2.02–4.185% and 16.63–18.38% respectively. The exudates are a good source of water soluble vitamins; ascorbic acid (7.04–26.40 mg 100g⁻¹), niacin (3.12–4.00 mg 100g⁻¹), riboflavin (0.14–0.54 mg 100g⁻¹) and thiamine (0.15–0.22 mg 100g⁻¹),). Both plants exudates are good sources of minerals such as Ca, Mg, P, Fe, Zn, Cu and Mn while Cr and Co were trace. These results indicate that exudates can be potential sources of feedstock for the pharmaceutical industry.     

Phage modification of the capacity of γ-irradiated Escherichia coli to support virus growth

The capacity of Escherichia coli to support the growth of T-even phages is more radiation resistant than it is to support the growth of T-odd phages. Since the differential effect is seen using the same irradiated cell culture, this effect must somehow be due to the characteristics of the phage.Here we have investigated the phospholipid metabolism in γ-irradiated E. coli B cells infected with T4 and T7 phage with the viewpoint that phospholipid synthesis, hence maintenance of membrane integrity, may be an essential factor in determining the capacity of cells to support phage growth. Results obtained using cells preirradiated with 100 krads show that the rate and amount of de novo phospholipid synthesis, as followed by ³²P labeling, in T4-infected cells are greater than in T7-infected cells. The rate continually increases from 0–15 min after infection, whereas in the latter cells there is a shut-off of synthesis beginning at around 10 min into infection. Using similarly irradiated cells, we found the course of turnover in T4-infected cells of phospholipids, prelabeled with [¹⁴C]acetate prior to infection, to show a pattern opposite that in T7-infected cells. The T4-infected cells show indications of redirection of synthesis, whereas the T7-infected cells show continued degradation starting at 5 min after infection. We conclude that there is a correlation between the ability of the phage to redirect the phospholipid metabolism of the cells, which ability the T4 phage is known to have, and the capacity of the irradiated cells to support a complete cycle of infection.The threshold γ-ray dose within which the capacity of E. coli B for supporting a normal plaquing efficiency of T4 is 300 krads, whereas that capacity for T7 declines exponentially with dose. However, within this dose range, the burst size and RNA synthesis of T4-infected cells decreases exponentially with dose, while those cells still surviving capacity loss for T7 growth give roughly normal bursts of this phage.The capacity of E. coli B for T4rII growth is similar in radiation resistance to that for T4r⁺ growth. The rate and amount of de novo phospholipid synthesis in rII-infected cells (where the cells were also irradiated with 100 krads prior to infection) are similar to those found in T4r⁺-infected cells for the first 10 min of infection, but thereafter diminishes in rate.     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures

The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the ⁺/^– state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.     

Blood platelets in human essential hypertension

Blood platelets of patients with essential hypertension display signs of both increased sensitivityin vitro to aggregating stimuli believed to contribute to thrombosis and of activationin vivo possibly expressing the release of vasoactive products. The mean features of the modified platelet profile in hypertension include an increased ₂-adrenergic receptor density, an enhanced rate of adhesion/aggregation in particular in response to ADP and arachidonic acid, a greater sensitivity for thrombin and adrenaline to stimulate increases in cytoplasmatic-free Ca²⁺, increased resting levels of cytoplasmatic-free Ca²⁺, a reduced content of serotonin often combined with a defective uptake mechanism, a facilitated efflux rate of noradrenaline, an exaggerated release reactionin vivo as indicated by the increased plasma levels of Betathromboglobulin and a shortened platelet life span. These changes occur to various extents in some, but not all, hypertensive patients and are not always strictly related to the degree of blood pressure increase. On the contrary, platelet cyclooxygenase and thromboxane synthetase activity are in the normal range.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.