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PubMed2006

Compositional assessment of event DAS-59122-7 maize using substantial equivalence.

Event DAS-59122-7 (Herculex RW) maize (Zea mays L.) plants were transformed to express the Cry34Ab1 and Cry35Ab1 binary insecticidal crystal proteins originally isolated from Bacillus thuringiensis Berliner (Bt) strain PS149B1. These proteins protect maize roots from attack by corn rootworms, Diabrotica spp. DAS-59122-7 maize also contains the pat gene, originally isolated from Streptomyces viridochromogenes, which confers tolerance to glufosinate-ammonium herbicides (e.g. Liberty). We assessed the composition of these transgenic plants (with and without Liberty herbicide treatment), grown at a total of eight fields sites over 2 years, by applying the principle of substantial equivalence. Forage and grain samples were analyzed for proximates, fiber and minerals, and grain was further analyzed for amino acids, fatty acids, vitamins, secondary metabolites and anti-nutrients. Data plots were prepared that allow for efficient investigation of equivalency between event DAS-59122-7 maize and a non-transgenic near-isogenic maize line grown contemporaneously. Results demonstrated that DAS-59122-7 maize is equivalent to non-transgenic maize with respect to these important constituents.

Amino Acids↗

PubMed1980

Factors inducing a loss of net negative surface charge on spleen cells of mice grafted with a slow-growing tumour.

The mean electrophoretic mobility (EPM) of splenic cells was determined in 4 different host-tumour systems. In splenic cells harvested from mice bearing slow-growing tumours, a significant EPM decrease was observed (12%) and in increase in the proportion of cells with slow mobility. Moreover, after 1 h incubation in RPMI medium at 37 degrees C and 2 washings, spleen cells showed a marked increase in their EPM (average 30%). Finally, the supernatant from incubation medium after contact with normal spleen cells (1 h at 37 degrees C) produced a significant decrease in their EPM (approximately 12%). On the other hand, no significant EPM variations were found between control spleen cells and cells from fast-growing tumours, before or after 1h incubation. The existence of factors which induce a loss of negative surface charge on spleen cells of some tumour-grafted mice is discussed.

Animals↗

PubMed1999

Tannic acid raises survival rate of mice bearing syngeneic tumors.

Tannic acid which was found earlier to have growth suppressive activity against cultured tumor cells, was given to Balb/c mice p.o. The mice had been inoculated with syngeneic tumor cells (Meth/A) i.p. When tannic acid was suspended in drinking water and given daily at doses of approximately 875 mg/kg/day and 1750 mg/kg/day, the respective survival rates were 59% and 48%, with that of the control mice being 29%. To study the cytotoxicity of tannic acid, we administered the tannic acid to mice at 875 and 1750 mg/kg/day for 35 days. The weight gain for each dose was lower than that of the control, although the difference was not significant. When tannic acid was given at 8750 mg/kg/day, the weight gain was significantly lower than the control. A histologic study did not show any pathological findings in the kidney, liver, or lungs in the mice given tannic acid at the above doses.

Administration, Oral↗

PubMed1998

Differential expression of two pro-opiomelanocortin mRNAs during temperature stress in common carp (Cyprinus carpio L.).

Pro-opiomelanocortin (POMC) is the precursor of a number of biologically active peptides, including adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and beta-endorphin, which are released by the pituitary glands of fish as well as mammals. To quantify the levels of expression of the two POMC mRNAs relative to one another during the response of the common carp to temperature-induced stress, we used reverse transcriptase PCR combined with capillary electrophoresis and laser-induced fluorescence detection. The ratio of POMC-I mRNA to POMC-II mRNA determined in wild-type and four isogenic carp strains was found to be strain-dependent and influenced by temperature. In strain E20xR8, the ratio had altered in favour of POMC-I from 1:3.2 (POMC-I:POMC-II) in fish adapted to 24 degreesC to 1:1.2 in fish adapted to a decrease of 9 degreesC in ambient temperature. A rapid drop in temperature from 24 to 15 degreesC decreased the POMC mRNA ratio at the expense of POMC-I from 1:1.9 in the control fish (strain E4xR3R8) to 1:4.2 3 h after the temperature drop of 9 degreesC. We conclude that both POMC genes are expressed in the common carp and that their expression ratio is strain-dependent and changes in response to ambient temperature.

Adaptation, Physiological↗

PubMed1979

Neonatal thymus grafts. II. Cellular events.

Neonatal thymus undergoes sequential necrosis and cell regeneration after grafting under the kidney capsule in normal and thymectomized recipients. This regeneration assessed in terms of cell number, response to mitogens, in vivo steroid sensitivity, level of antigen-binding cells, and cellular distribution on Ficoll gradient is not influenced by the presence of host thymus or by FTS injections. The grafted thymus shows a similar pattern of development as control thymus of the same age followed up in situ, with the exception of age-related involution the onset of which takes place earlier in the grafted thymus.

Animals↗

PubMed1979

Induction and characterization of isogeneic anti-idiotypic antibodies to BALB/c myeloma S117: lack of reactivity with major idiotypic determinants.

Isogeneic anti-idiotypic antibodies were induced by immunization of BALB/c mice with the BALB/c-derived myeloma protein S117 which binds N-acetylglucosamine-containing antigens including Group A streptococcal carbohydrate (A-CHO). Most BALB/c mice produced anti-S 117 idiotypic antibodies, as shown by various different immunization protocols. The antibodies of individual mice were of intermediate to high affinity (2.8 x 10(6) M-1 to 1.4 x 10(8) M-1). In isoelectric focusing, most individual antibodies were shown to consist of a small number of clonotypes, but each mouse produced its own unique set of clones so that the potential clonal repertoire of strain BALB/c is rather large. Most importantly, all isogeneic anti-idiotypic antibodies were directed against idiotypic determinants of S117 that are not shared with induced anti-A-CHO antibodies, whereas it has been shown previously that allogeneic and xenogeneic anti-S 117 idiotypic antibodies react with idiotypic determinants unique to S 117 as well as with those that are shared with anti-A-CHO antibodies. The data suggest that the expression of S 117 idiotypic determinants in strain BALB/c is not under antibody-mediated, anti-idiotypic feedback control.

Acetylglucosamine↗

PubMed2003

Studies on the expression of regulatory locus sae in Staphylococcus aureus.

Global regulatory locus sae consists of a two-component signal transduction system coded by saeR and saeS genes that upregulates the transcription of several exoproteins. Northern analysis carried out in this study reveals the synthesis at late and post-exponential phases of a cotranscript of saeR and saeS structural genes of about 2.4 kb. This transcript is diminished in the isogenic agr:: tetM mutant. Likewise, transcriptional fusion experiments show that sae expression is downregulated in the agr null mutant. Complementation analyses with plasmids carrying fragments of about 1.2 or 0.2 kbp upstream of saeR-saeS genes, which restore fully or only partially, respectively, the wild-type phenotype to the sae mutant, are in agreement with two initiation start points of transcription revealed by primer extension experiments. This work, as well as previous studies, reveals a complex hierarchical regulatory network involving several loci that control the expression of virulence determinants in S. aureus.

Bacterial Proteins↗

PubMed1992

Authentic temperature-regulation of a heat shock gene inserted into yeast on a high copy number vector. Influences of overexpression of HSP90 protein on high temperature growth and thermotolerance.

Heat shock protein HSP90 is relatively abundant in eukaryotic cells even in the absence of heat shock. Its precise function is still unclear, although it is apparently required in higher levels for growth at high temperatures. In this study Saccharomyces cerevisiae transformants were constructed with 50-150 copies of the homologous heat-inducible gene for HSP90 (HSP82) present on a high copy number episomal vector. These transformants were then used to demonstrate: (i) that this heat shock gene displays essentially normal regulation when present in yeast at high copy numbers; (ii) that yeast is an expression host suitable for the high level synthesis of HSP90; and (iii) that increasing normal cellular levels of HSP90 affects a number of physiological properties. The HSP82 gene is normally single-copy in the haploid yeast genome, yet even at 50 to 150 copies per cell it displayed almost normal basal and heat shock-induced levels of expression. Proper regulation of the heat shock element sequence controlling HSP82 is therefore not lost at high gene copy levels. In unstressed cultures in exponential growth at 25 degrees C the low basal expression of the multiple HSP82 gene copies caused a 3 to 7-fold HSP90 overproduction, but HSP90 levels increased 10-fold to 30-40% of total cell protein following temperature upshift to 39 degrees C for 75 min. Heat induction of the chromosomal genes for other heat shock proteins in the same cells was not suppressed relative to cells which were isogenic but for the possession of just a single HSP82 gene, this constituting further evidence that yeast can authentically regulate a large number of heat shock genes. HSP90 overproduction was not protective against heat killing, causing strain-dependent reductions in growth at 37.5 degrees C and in thermotolerance.

Autoradiography↗

PubMed2004

How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis.

The optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels.

Carbohydrate Metabolism↗

PubMed2002

Assessment of protective efficacy of live and killed vaccines based on a non-encapsulated mutant of Streptococcus suis serotype 2.

The protective efficacy of a live and killed non-encapsulated isogenic mutant of Streptococcus suis serotype 2 was determined in pigs, and compared with the efficacy of the capsulated wild-type strain. SPF pigs were vaccinated twice intramuscularly at 4 and 7 weeks of age with a dose of 1 x 10(9) formalin-killed CFU of the wild-type (WT-BAC), formalin-killed non-encapsulated mutant (CM-BAC) or live non-encapsulated mutant (CM-LIVE) strain. After 2 weeks, vaccinated pigs and non-vaccinated controls were challenged intravenously with 1 x 10(7) CFU of the homologous, wild-type S. suis serotype 2 strain. Protection was evaluated by clinical, bacteriological, serological and post-mortem examinations. All pigs vaccinated with WT-BAC were completely protected against challenge with the homologous serotype. Pigs vaccinated with CM-BAC were partially protected. Although all pigs vaccinated with CM-BAC survived the challenge, four out of five pigs developed clinical signs of disease for several days. Compared to the WT-BAC and CM-BAC, the CM-LIVE vaccine was less protective. Two out of five pigs vaccinated with CM-LIVE died in the course of the experiment and all of them developed specific clinical signs of disease for several days. The protective efficacy of the vaccines could be associated with serum antibody titers. Antibody titers against cells of wild-type and non-encapsulated mutant strains as well as against muramidase-released proteins (MRP) were high in pigs vaccinated with WT-BAC and CM-BAC. Pigs vaccinated with CM-LIVE showed lower antibody titers. Antibody titers against purified capsular polysaccharides (CPS) of S. suis serotype 2 were only found in pigs vaccinated with WT-BAC. These findings indicate that CPS and other bacterial components of WT-BAC are probably essential for full protection against homologous challenge.

Animals↗

PubMed1998

Analysis of natural allelic variation at flowering time loci in the Landsberg erecta and Cape Verde Islands ecotypes of Arabidopsis thaliana.

We have analyzed the flowering behavior of two Arabidopsis ecotypes: the laboratory strain Landsberg erecta (Ler) and an ecotype from the tropical Cape Verde Islands (Cvi). They differ little in their flowering phenotypes and in their responses to photoperiod length changes and to vernalization treatment. However, segregating populations derived from crosses between them showed a much larger variation. An approach of quantitative trait locus (QTL) mapping in recombinant inbred lines (RILs) grown under three environments differing in day-length and/or vernalization treatment has been used to detect and locate flowering loci. Four main QTLs were identified, designated early day-length insensitive (EDI), flowering F, G, and H (FLF, FLG, and FLH, respectively), to which most of the flowering behavior differences could be attributed. To further characterize the individual loci, near isogenic lines were constructed by introgressing Cvi early alleles of EDI and FLH into the Ler genetic background. EDI-Cvi alleles produce earliness under both long- and short-day photoperiods, rendering Ler plants almost day-length neutral. In addition, RILs were selected to analyze FLF and FLG. These loci interact epistatically and RILs carrying late alleles at FLF and FLG were very responsive to vernalization and showed an increased response to photoperiod length changes. The possible role of these loci for the control of flowering is discussed in the context of the current Arabidopsis model.

Alleles↗

PubMed2002

Attenuation of the virulence of Porphyromonas gingivalis by using a specific synthetic Kgp protease inhibitor.

The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.

Adhesins, Bacterial↗

PubMed2005

Glycolytic enzyme activities are decreased during acute rejection in transplanted rat hearts.

Metabolic alterations have been characterized in various heart diseases. However, no data are available concerning metabolic changes during acute rejection episodes. Heterotopic heart transplantations in rats were done using Lewis rats as donors and recipients as a control group. The rejection group included Brown-Norway rat donors to Lewis rat recipients. Nonoperated hearts were also studied. Enzyme activities were determined for phosphofructokinase, pyruvate kinase, and lactate dehydrogenase. There were no alterations in the control group compared to nonoperated hearts. However, the rejection cohort of hearts showed decreased glycolytic enzymes. Although lactate dehydrogenase maintained similar levels compared to the control group, phosphofructokinase showed only 50% activity, and pyruvate kinase showed less than 10% of the activity compared with controls. These results suggested that metabolic alterations in rejected hearts differ from other cardiomyopathies.

Acute Disease↗

PubMed2006

Early short-term platelet-derived growth factor inhibition prevents the development of chronic allograft nephropathy in experimental rat kidney transplantation.

Chronic allograft nephropathy (CAN) remains the primary reason for late allograft loss in kidney transplantation. Platelet-derived growth factor (PDGF) is a major mitogen mediating mesenchymal cell proliferation in CAN. When administered continuously the PDGF receptor tyrosine kinase inhibitor imatinib prevents the development of CAN and restores kidney function in experimental kidney transplantation. Herein we investigated whether early short-term imatinib treatment prevented CAN. Kidney transplantations were performed from DA to WF rats and syngenic controls were done between DA rats. Allograft recipients were immunosuppressed with cyclosporine (CsA; 1.5 mg/kg/d sc). One group of allografts was also treated with imatinib (10 mg/kg/d po). Serum creatinine levels were measured once a week. Grafts were harvested 90 days after transplantation for histology and immunohistochemistry (PDGF-AA, -BB, PDGFR-alpha, -beta). Histological changes were scored according to the Chronic Allograft Damage Index (CADI). Among syngenic grafts, no signs of CAN were observed, namely, CADI 0.3 +/- 0.2 (mean +/- SEM). Control allografts showed moderate to intense chronic changes, CADI 6.5 +/- 1.3. Early short-term imatinib treatment significantly prevented the development of CAN compared with control allografts. Only a few histological changes were seen, namely, CADI 3.3 +/- 1.4. Compared with control allografts PDGF ligand and receptor induction was significantly inhibited by imatinib to nearly the same level as in syngenic grafts. Creatinine values of imatinib-treated allografts were also lower than control allografts. Our results demonstrated that early short-term imatinib treatment significantly prevented CAN. This indicated that early PDGF induction has an important role in the pathogenesis of CAN.

Animals↗

PubMed1977

Quantitative in vivo studies of soluble simian virus 40 tumor-specific transplantation antigens of the mouse.

Quantitative studies have been performed on the immunogenicity of a membrane-bound antigen of a simian virus 40 (SV40) -induced sarcoma in syngeneic BALB/c mice and of subcellular fractions derived from this tumor. The objectives of the investigation were: a) to develop a quantitative in vivo assay of the tumor-specific transplantation antigen (TSTA) and b) to compare the distribution of histocompatibility antigens, H-2, with that of the SV40 TSTA during several fractionation steps. The immunogenicity of the TSTA-containing fractions was assessed from dose-response curves relating tumor size and the amount of protein used for immunization. After digestion of the tumor cell membranes with a limited amount of papain, H-2 as well as TSTA were present in a soluble form. A single immunization with only 2 microng of the solubilized TSTA reduced the tumor size by 70% compared to that in nonimmunized control animals. The results of several fractionation steps suggest that H-2 and the TSTA are not tightly associated in the solubilized immunogenic material.

Animals↗

PubMed2004

Exercise induces hypoglycemia in rats with islet transplantation.

Recently, islet transplantation in patients with type 1 diabetes has had greater success than in the past, but the important question of whether the kinetics of islet secretion are able to accommodate the metabolic demands of special conditions such as exercise remains unanswered. Syngeneic rat islets (4,000 islet equivalents/rat) were transplanted into the liver, kidney, and peritoneal cavity (encapsulated or nonencapsulated) of rats with streptozocin-induced diabetes. Normoglycemic transplanted rats and age-matched controls were subjected to 30 min of moderate exercise on a treadmill 5 weeks after transplantation. Although control rats maintained near normoglycemia during and after exercise, the rats with islet transplants had significantly lower blood glucose levels. For the rats with islets in the liver, increased C-peptide levels were found at 30 min (790 +/- 125 and 1,450 +/- 250 pmol/l at 0 and 30 min, respectively; P < 0.01), whereas a decrease was found in controls and in rats with islets transplanted into the peritoneal cavity or under the kidney capsule. Moreover, increased glucagon levels were found after exercise in the rats with islets transplanted into the liver (62 +/- 6, 165 +/- 29, 155 +/- 27, and 97 +/- 13 pg/ml at 0, 30, 60, and 90 min, respectively; P < 0.05), whereas no changes in glucagon levels were observed in controls. In conclusion, moderate exercise caused hypoglycemia in rats with islet transplants in different sites including liver, kidney, and peritoneal cavity. C-peptide and glucagon responses to exercise were very different in rats with transplanted islets compared with controls. This islet dysfunction led to exercise-induced hypoglycemia.

Animals↗

PubMed1978

Transplantation of the "presumptive" Peyer's patch of fetus or newborn mice under the kidney capsule of adult syngeneic recipient.

Small fragments of caecum from fetus, newborn or several-day-old mice were transplanted under the kidney capsule of adult syngeneic hosts. Transplants were derived from such a part of caecum at which in adult mice a Peyer's patch is always present (experimental group) or from such a part at which Peyer's patch is never located (control group). Donors were chosen from such early developmental stage at which Peyer's patches not yet been formed, hoping to observe Peyer's patch formation in transplants in view of possible influence of intestinal epithelium on a lymphocyte homing. After 10 to 150 days, transplants were asessed histologically. None Peyer's patche was formed. However, small accumulations of lymphoid cells were located in vicinity of epithelial cysts and blood vessels occasionally filled with lymphocytes were seen in the experimental but not in control group. The results showed that either number of epithelial cells or degree of their specialization were unsufficient for Peyer's patch formation. Other factors like the antigenic stimulation from the gut lumen might be thus necessary for Peyer's patch development.

Animals↗

PubMed1996

A direct comparison of approaches for increasing carbon flow to aromatic biosynthesis in Escherichia coli.

Different approaches to increasing carbon commitment to aromatic amino acid biosynthesis were compared in isogenic strains of Escherichia coli. In a strain having a wild-type PEP:glucose phosphotransferase (PTS) system, inactivation of the genes encoding pyruvate kinase (pykA and pykF) resulted in a 3.4 fold increase in carbon flow to aromatic biosynthesis. In a strain already having increased carbon flow to aromatics by virtue of overexpression of the tktA gene (encoding transketolase), the pykA and/or pykf mutations had no effect. A PTS- glucose+ mutant showed a 1.6-fold increase in carbon flow to aromatics compared to the PTS+ control strain. In the PTS- glucose+ host background, overexpression of tktA caused a further 3.7-fold increase in carbon flow, while inactivation of pykA and pykF caused a 5.8-fold increase. When all of the variables tested (PTS-glucose+, pykA, pykF, and overexpressed tktA) were combined in a single strain, a 19.9-fold increase in carbon commitment to aromatic biosynthesis was achieved.

Amino Acids↗