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PubMed1998

Production and selection of antigen-specific fully human monoclonal antibodies from mice engineered with human Ig loci.

The ability to produce highly specific fully human monoclonal antibodies to human antigens has potential significant applications to human therapy. This review describes the creation of novel mouse strains engineered to produce a diverse repertoire of fully human antibodies in the absence of mouse antibodies. These mouse strains have been generated by introducing megabase-sized human immunoglobulin loci, containing the majority of the human antibody gene repertoire, in nearly germline configuration, into mice deficient in mouse antibody production. The mice produce high levels of human IgMkappa and IgGkappa antibodies with a diverse adult-like repertoire. Upon immunization with multiple human antigens the mice generate high affinity, antigen-specific fully human monoclonal antibodies with neutralization activity. Comparison of these mice to other strains containing limited human antibody gene repertoire underscores the importance of the large number of variable genes for faithful reproduction of functional and diverse human antibody response in mice.

Journal Article↗

PubMed1995

Pore strain behaviour of collagen-glycosaminoglycan analogues of extracellular matrix.

Mechanical properties of a highly porous collagen-glycosaminoglycan (CG) analogue of extracellular matrix were investigated. The stress-strain behaviour of the matrices displayed the typical concave-up shape characteristic of porous materials and soft tissues. Deformation behaviour of the material was further investigated using novel methodology to determine material strains at the micrometre level. Central to the methodology was the use of scanning electron microscopy (SEM) in conjunction with a digital image analyser. The method for strain determination at the micrometre level was verified by comparison with measurement of surface strain using an optical method. An unexpected finding in these materials is that Poisson's ratio varies with engineering strain. A geometrical/mathematical model was developed to explain this behaviour. These results encourage further investigation of the mechanical properties of these materials, in order to provide important insights into the physical microenvironment in which cells reside.

Biocompatible Materials↗

PubMed1996

The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane.

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high-copy-number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild-type strain with respect to YopB-dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB-dependent translocation mechanism from surface-located bacteria and subsequently targeted to the inner surface of the target-cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.

Bacterial Outer Membrane Proteins↗

PubMed1996

The listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C synergize to elicit endothelial cell phosphoinositide metabolism.

Exotoxins such as listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PIcA) have been implicated in listerial infection and sepsis. Employing different Listeria strains, mutated in individually known virulence genes, we examined exotoxin-related induction of endothelial cell signaling. Listeria monocytogenes was a potent inductor of phosphatidylinositol (PtdIns) metabolism in HUVEC. This effect was completely absent in a LLO-negative strain. Using a recombinant Listeria innocua strain, engineered to produce high levels of LLO, PtdIns metabolism was restored to approximately 30% of that produced by the parental L. monocytogenes strain. A recombinant L. innocua strain expressing only PIcA did not induce any PtdIns metabolism. Even higher than wild-type levels of PtdIns hydrolysis products were, however, evoked when engineered bacteria secreted both LLO and PIcA. These effects occurred in the absence of bacterial uptake by the endothelial cells. Corresponding results were observed with regard to endothelial diacylglycerol (DAG) generation. The amplification of endothelial cell signaling could be reproduced by engaging purified LLO and PIcA in the absence of bacteria. In these experiments, the unrelated pore-forming agent staphylococcal alpha-toxin, a very weak stimulus for endothelial phosphoinositide metabolism by itself, substituted for LLO to allow marked PtdIns hydrolysis when co-applied with PIcA. We conclude that the listerial exotoxins LLO and PIcA cooperate to provoke potent second messenger synthesis in endothelial cells, in the absence of cell invasion by the bacteria. This is an impressive example of synergism between a pore-forming and an enzymatic bacterial exotoxin in provoking cell signaling and inflammatory events.

Bacterial Proteins↗

PubMed2003

Diversity of oligopeptide transport specificity in Lactococcus lactis species. A tool to unravel the role of OppA in uptake specificity.

The specific oligopeptide transport system Opp is essential for growth of Lactococcus lactis in milk. We examined the biodiversity of oligopeptide transport specificity in the L. lactis species. Six strains were tested for (i) consumption of peptides during growth in a chemically defined medium and (ii) their ability to transport these peptides. Each strain demonstrated some specific preferences for peptide utilization, which matched the specificity of peptide transport. Sequencing of the binding protein OppA in some strains revealed minor differences at the amino acid level. The differences in specificity were used as a tool to unravel the role of the binding protein in transport specificity. The genes encoding OppA in four strains were cloned and expressed in L. lactis MG1363 deleted for its oppA gene. The substrate specificity of these engineered strains was found to be similar to that of the L. lactis MG1363 parental strain, whichever oppA gene was expressed. In situ binding experiments demonstrated the ability of OppA to interact with non-transported peptides. Taken together, these results provide evidence for a new concept. Despite that fact that OppA is essential for peptide transport, it is not the (main) determinant of peptide transport specificity in L. lactis.

ATP-Binding Cassette Transporters↗

PubMed2000

Strain-specific restriction of the antiphagocytic property of group A streptococcal M proteins.

Group A streptococcal M proteins are type-specific virulence factors that inhibit phagocytosis. We used two M proteins, M5 and Emm22, to analyze the influence of genetic background on the properties of M proteins. Mutant strains, engineered to lack these M proteins, were complemented with genes encoding the homologous or heterologous M protein, and the complemented strains were analyzed for phagocytosis resistance. Neither the M5 nor the Emm22 protein conferred phagocytosis resistance in the heterologous background, but they did do so in the homologous background. This was not due to lack of surface expression in the heterologous background. Moreover, the M5 and Emm22 proteins expressed in heterologous background appeared to have normal structure, since they were not affected in their ability to bind different human plasma proteins. In particular, M5 or Emm22 had normal ability to bind human complement inhibitors, a property that has been implicated in phagocytosis resistance. Results similar to those obtained with M5 and Emm22 were obtained in experiments with the M6 and Emm4 proteins. Together, these data suggest that the surface expression of M protein alone may not be sufficient to confer phagocytosis resistance and consequently that strain-specific factors other than M and Emm proteins may contribute to the ability of group A streptococci to resist phagocytosis.

Antigens, Bacterial↗

PubMed2004

Enhanced expression of insecticidal crystal proteins in wild Bacillus thuringiensis strains by a heterogeneous protein P20.

P20 is a small chaperone-like protein encoded by a cry11A operon in Bacillus thuringiensis subsp. israelensis (Bti); it is essential to Cyt1Aa expression. In this report, the gene P20 was transformed into wild strains of subsp. kurstaki to raise the yield of crystal proteins. As a result, larger crystals were produced by the transformant than by the wild control, and most were in the form of a big bipyramid (average 2.4 microm long); some were irregular because of too high expression, while the spores turned out to be small spheroids unlike the long rods in the wild strains. SDS-PAGE analysis confirmed that Cry1A protoxin production was doubled by P20, but no increase of Cry2A production was observed. Besides, P20 caused obvious changes not only in bacterial morphology, but in the sporulation process as well. Further investigation discovered that a serious degradation happened to Cry1A in vivo of the wild strains, and reconfirmed that P20 was effective in preventing the degradation. Our results suggest that P20 is useful in engineered strain construction with enhanced protein expression.

Bacillus thuringiensis↗

PubMed1997

Recent advances in the construction of bacterial genotoxicity assays.

Bacterial mutagenicity assays have been widely used in genotoxicology research for two decades. We discuss the development of such assays, especially the Ames test, with particular attention to strain engineering. Genes encoding enzymes of mutagen bioactivation, including N-acetyltransferase, nitroreductase, and cytochrome P450, have been introduced into tester strains. The processing of DNA damage by the bacterial strains has also been modified in several ways, so as to enhance mutagenesis. These efforts have greatly increased the sensitivity of mutation assays and have illuminated the molecular mechanisms of mutagenesis. We also discuss the relationship between bacterial assays and in vivo mutation assays which use transgenic rodents.

Air Pollutants↗

PubMed2004

Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of sulfotransferases and acetyltransferases in nitrofen mutagenicity.

Nitrofen had been used as a herbicide, until its carcinogenic and teratogenic activity in rodents was detected. A food contamination occurring in 2002 in Germany led to the initiation of new studies in order to better understand the potential risk for humans. Nitrofen is a nitroarene and as such might be activated to a mutagen via reduction to the corresponding hydroxylamine and subsequent formation of a reactive acetic or sulfuric acid ester. Therefore, we have investigated the mutagenicity of nitrofen in Salmonella typhimurium strains engineered for the expression of all human xenobiotic-metabolizing sulfotransferases (SULTs) and acetyltransferases (NATs) identified. Nitrofen was inactive in the parental strains TA1538, TA98 and TA100, but was mutagenic even at low doses when human sulfotransferase SULT1A1 (the major broad-spectrum phenol SULT) was expressed in these strains, but not when it was expressed in a TA1538-derived strain deficient in an endogenous nitroreductase. Several other human SULTs (in particular 1A3 and 1C1) as well as human NAT2 (unlike NAT1) also activated nitrofen, but were markedly less efficient than SULT1A1. Likewise, expression of rat and mouse SULT1A1 led to weaker mutagenic activity of nitrofen than expression of the corresponding human enzyme. An endogenous acetyltransferase only activated nitrofen to a mutagen when it was strongly over-expressed in the TA98-derived strain YG1024. Thus, humans might be more susceptible to the carcinogenic effects of nitrofen than mice and rats, which have been used in long-term studies. The fact that several SULTs show particular high expression in fetal tissues suggests that this activation pathway may also play a role in the teratogenic effects observed.

Animals↗

PubMed1987

Residual stress and strain in aortic segments.

In the study of stresses and strains in vascular segments, it is generally assumed that the traction-free configuration assumed by a segment when there is no axial force and there are no intravascular and extravascular pressures is stress-free. To investigate the degree of validity of this assumption, 286 oval shaped rings were excised from three bovine and six porcine aortas and photographed. Radial cuts were made in these rings which opened up into horseshoe shapes and were also photographed. Smoothed boundary lengths at intimal and adventitial levels in the rings and their cut open configurations were measured from the photographs and the residual strains in the annular configuration relative to the open configuration were computed. It was found that: the average maximum residual intimal engineering strain in the uncut configuration was -0.082 for all nine aortas and -0.096 and -0.077 for the bovine and porcine aortas alone, respectively; the average maximum residual adventitial strain was 0.085 for all aortas, and 0.102 and 0.078 for the bovine and porcine aortas alone, respectively; an estimated average beneficial compressive stress of -0.188 X 10(5) Pa (corresponding to a strain level of -0.082) is available at the intimal level to counteract the in vivo tensile stress due to the intravascular pressure; an estimated average initial tensile stress of 0.195 X 10(5) Pa (corresponding to a strain level of 0.085) exists at the adventitial level which adds to the in vivo tensile stress due to the intravascular pressure. Although these stress levels are not large in comparison with the in vivo stress in the arterial wall, a detailed stress analysis must take into account these initial stresses.

Animals↗

PubMed1997

Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk.

The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The reduction in proteolytic activity of the PI-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the PIII-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the PIII-type proteinase strain.

Animals↗

PubMed2007

Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain.

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with a Cryptococcus neoformans strain engineered to produce the Th1-type cytokine gamma interferon (IFN-gamma). Mice given a pulmonary infection with an IFN-gamma-producing C. neoformans strain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenic C. neoformans strain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with a C. neoformans strain expressing IFN-gamma results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.

Animals↗

PubMed1997

Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase.

The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1delta mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production ( x 4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production.

Base Sequence↗

PubMed1986

Dynamic performance characteristics of the liquid metal strain gage.

Performance characteristics of the liquid metal strain gage (LMSG) were evaluated by both static and dynamic bench testing. Statically, the devices were found to have outputs closely proportional to engineering strains, up to strain levels of 40%. While individual gage factors varied appreciably (up to 50%), each of the gages studied showed excellent reproducibility of behavior. Dynamically, the response to sinusoidal strain inputs was frequency-independent up to 50 Hz, and there was no detectable phase shift. Similarly, the LMSG response to constant-speed displacement inputs was velocity-independent over the range of nominal strain rates from 20 s-1 to 0.02 s-1. The devices proved capable of maintaining stable outputs when held stretched to fixed lengths, even if such tests were performed immediately following stepwise displacement inputs. Thermal artifacts were found to be modest (0.185% apparent strain per degree C), and there was no appreciable sensitivity to non-axial strains. When mounted on an in vitro ligament preparation, the LMSG measured apparent ligament strain similar to that detected by a video dimension analyzer. A protocol by which an implanted LMSG could be used to infer in vivo muscle forces was demonstrated, based on recordings of tendo-Achilles strains developed by a rabbit during slow hopping.

Animals↗

PubMed2002

Re-engineering the sterile insect technique.

The mass release of sterile insects (the Sterile Insect Technique, SIT) is a highly effective area-wide method of pest control with a low environmental impact. SIT relies on the sterilization by irradiation of large numbers of insects. This has unavoidable costs in terms of the fitness of the irradiated insects and the financial requirements of constructing and operating the radiation facility. In many cases it is considered important to release only males, but large-scale sex-separation is also problematic. I have proposed that both of these difficulties can be overcome by using engineered strains of insects carrying a dominant, repressible, lethal gene or genetic system. As a proof of principle, my group and others have constructed strains of Drosophila melanogaster with the required genetic properties.

Animals↗

PubMed2005

Construction of transgenic Bacillus mucilaginosus strain with improved phytase secretion.

AIMS: To construct a transgenic Bacillus mucilaginosus strain to increase the secretion capability of a wild-type isolate of B. mucilaginosus D4B1 to hydrolyse phytate phosphorus, which can be used as a microbial fertilizer in field application. METHODS AND RESULTS: We constructed a phytase secreting expression vector pSP43 with a mini-Tn5 transposon and a Aspergillus fumigatus phytase expression cassette. The vector pSP43 was successfully transferred into the wild-type B. mucilaginosus using the particle bombardment method, and three transgenic strains with a stable copy of phytase expression cassette integrated into the chromosome of the B. mucilaginosus by Tn5 transposition were selected. The phytase activity of the engineered strains increased 36-46-fold when compared with the wild-type strain of D4B1. CONCLUSIONS: The A. fumigatus phytase gene can be expressed under the direction of p43 promoter in B. mucilaginosus. The expression protein is secreted extracellularly and newly constructed strains showed a high phytase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: A transgenic Bacillus strain by the particle bombardment method was constructed.

6-Phytase↗

PubMed1999

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase.

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.

Acetates↗

PubMed1992

Monitoring of Yersinia enterocolitica in murine and bovine feces on the basis of the chromosomally integrated luxAB marker gene.

We previously integrated the luxAB gene into the Yersinia enterocolitica chromosome. In this article, we assessed, by luminometry, the survival of the engineered strain KNG1024 in the digestive tracts of mice and cows. In situ detection and a count of the released strain were performed on feces from orally inoculated BALB/c mice for 24 days. This method is a rapid and reliable system for long-term monitoring of genetically engineered bacteria. In cow feces, the count of Y. enterocolitica ranged from 210 to 6,000 CFU/g of feces. This very low count was not detectable by direct luminometry.

Animals↗