Investigation of bacterial nucleotide excision repair using single-molecule techniques

Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER.     

Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.

An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.     

Differential excision of carcinogenic hydrocarbon-DNA adducts in mouse embryo cell cultures.

Primary mouse embryo cell cultures efficiently excise DNA damage introduced by the carcinogens 7-bromomethylbenz[$\text{a}$]anthracene and 3-methylcholanthrene but are inefficient in excision of damage introduced by 7,12-dimethylbenz[a]anthracene. Since exposure of the cells to the latter compound does not impair their capacity for excision of adducts introduced by the bromocompound, it is concluded the 7,12-dimethylbenz[$\text{a}$]anthracene-DNA adducts are intrinsically difficult to excise.     

On-line monitoring of recombinant bacterial cultures using multi-wavelength fluorescence spectroscopy

Multi-wavelength fluorescence spectroscopy was evaluated as a tool for on-line monitoring of recombinant Escherichia coli cultivations expressing human basic fibroblast growth factor (hFGF-2). The data sets for the various combinations of the excitation and emission spectra from batch cultivations were analyzed using principal component analysis. Chemometric models (the partial least squares method) were developed for correlating the fluorescence data and the experimentally measured variables such as the biomass and glucose concentrations as well as the carbon dioxide production rate. Excellent correlations were obtained for these variables for the calibration cultivations. The predictability of these models was further tested in batch and fed-batch cultivations. The batch cultivations were well predicted by the PLS models for biomass, glucose concentrations and carbon dioxide production rate (RMSEPs were respectively 5%, 7%, 9%). However, when tested for biomass concentrations in fed-batch cultivations (with final biomass three times higher than the highest calibration data) the models had good predictability at high growth rates (RMSEPs were 3% and 4%, respectively for uninduced and induced fed-batch cultivations), which was as good as for the batch cultivations used for developing the models (RMSEPs were 3% and 5%, respectively for uninduced and induced batch cultivations). The fed-batch cultivations performed at low growth rates exhibited much higher fluorescence for fluorophores such as flavin and NAD(P)H as compared to fed-batch cultivations at high growth rate. Therefore, the PLS models tended to over-predict the biomass concentrations at low growth rates. Obviously the cells changed their concentration of biogenic fluorophores depending on the growth rate. Although multi-wavelength fluorescence spectroscopy is a valuable tool for on-line monitoring of bioprocess, care must be taken to re-calibrate the PLS models at different growth rates to improve the accuracy of predictions.     

Temperature dependent alteration in bacterial protein composition

When different strains of $\text{Escherichia coli}$ or $\text{Salmonella}$ are grown at 30° or at 42° and their proteins are compared using SDS-polyacrylamide gel electrophoresis, differences are observed in the protein composition of the cells. In particular, one major difference is in a protein of molecular weight of approximately 58,000, which is made at 42° but not at 30°.     

Specific induction of secondary product formation in transgenic plant cell cultures using an inducible promoter

This paper describes the artificial induction of secondary metabolite production in transgenic plant cell cultures using a recombinant, inducible plant promoter. The bacterial gene ubiC from Escherichia coli encodes the enzyme chorismate pyruvate lyase (CPL) which catalyses the conversion of chorismate to 4-hydroxybenzoate (4HB). This gene was fused to the tetracycline-inducible plant promoter Triple-Op. After transformation into Nicotiana tabacum W38 TET, transgenic cell cultures were established. Addition of chlorotetracycline to the medium led to specific induction of CPL activity. The optimal chlorotetracycline concentration was approximately 2 mg/l medium. Three to 5 h after induction, the ubiC mRNA concentration reached a maximum, while highest specific CPL activity was detected after 8 days. The artificial secondary metabolite 4HB was converted to glucosides, and their accumulation reached maximum levels after 5 weeks of subculture. The induction was reversible. Received: 31 May 1997 / Revision received: 22 August 1997 / Accepted: 30 September 1997     

Efficient induction of ginsenoside biosynthesis and alteration of ginsenoside heterogeneity in cell cultures of Panax notoginseng by using chemically synthesized 2-hydroxyethyl jasmonate

Chemically synthesized 2-hydroxyethyl jasmonate (HEJA) was for the first time employed to induce the ginsenoside biosynthesis and to manipulate the product heterogeneity in plant cell cultures. The dose response and timing of HEJA elicitation were investigated in cell suspension cultures of Panax notoginseng. The optimal concentration and timing of HEJA addition for both cell growth and ginsenoside accumulation was identified to be 200 μM added on day 4. It was interestingly found that HEJA could stimulate ginsenosides biosynthesis and change their heterogeneity more efficiently than methyl jasmonate (MJA), i.e., the total ginsenoside content and the Rb/Rg ratio increased about 60 and 30% with HEJA elicitation than that by MJA, respectively. The activity of Rb₁ biosynthetic enzyme, i.e., UDPG-ginsenoside Rd glucosyltransferase (UGRdGT), was also higher in the former case. A maximal production titer of ginsenoside Rg₁, Re, Rb₁, and Rd was 47.4±4.8, 52.3±4.4, 190±18, and 12.1±2.5 mg/l with HEJA elicitation, which was about 1.3-, 1.3-, 1.7-, and 2.1-fold than that using MJA, respectively. Early signal events in plant defense response, including oxidative burst and jasmonic acid (JA) biosynthesis, were also examined. Levels of H₂O₂ and NO in medium and l-phenylalanine ammonia lyase activity in cells were not affected by addition of MJA and HEJA. On the other hand, the JA content in cells was increased with external jasmonates elicitation, and it was inhibited with the addition of JA biosynthesis inhibitors. The results suggest that oxidative burst might not be involved in the jasmonates-elicited signal transduction pathway, and MJA and HEJA may induce the ginsenoside biosynthesis via induction of endogenous JA biosynthesis and key enzymes (such as UGRdGT) in the ginsenoside biosynthetic pathway of P. notoginseng cells. The information is useful for hyperproduction of plant-specific heterogeneous products.     

Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.     

Efficient bacterial production of functional antibody fragments using a phagemid vector

The so-called ‘in vitro evolutionary method’ using a phage display system has been applied for protein engineering of the antigen-binding fragment of antibodies (Fab) by conducting random mutagenesis at the antigen-binding site in combination with antigen-based biopanning. However, isolated phage clones displaying Fab cannot necessarily be used for efficient bacterial production of engineered Fab proteins, often due to deleterious defects in their proper folding abilities derived in compensation for the gain of high affinity for a particular antigen. We here report a new method of an efficient and direct bacterial expression system for the phagemid-coded Fab proteins without use of the helper phage. To overcome a low folding efficiency derived from somatic hypermutations, if any, we have established optimum conditions for bacterial cultivation and protein expression, utilizing unusually long cultivation time (>50 h) and very low temperature (25 °C) and thereby leading to the production and extracellular secretion of Fab proteins in a very high yield (3–15 mg/L of culture). The purified Fab folded correctly and could efficiently bind an antigen, as judged by circular dichroism and isothermal titration calorimetry, respectively.     

Platelet stimulation for prostacyclin production in aortic endothelial cell cultures: alteration in diabetes mellitus.

We evaluated the effect of platelets on prostacyclin (PGI2) production in bovine aortic endothelial cell cultures. Human platelet extract significantly stimulated PGI2 production by cultured aortic endothelial cells in a time- and dose-dependent manner, suggesting that platelets contain PGI2-stimulatory activity (PSA). Supernatant fluid separated from platelets activated by collagen also exhibited PSA. The factor(s) causing the PSA of platelets was non-dialysable and heat-stable (56 degrees C for 30 min or 100 degrees C for 3 min), was completely inhibited by trypsin pretreatment, and exhibited an affinity to heparin agarose. Furthermore, gel filtration chromatography showed that the factor(s) responsible for the platelet PSA was eluted at three different peaks with approximate molecular weights of 50,000, 25,000 and 11,000. The PSA of platelet extract from patients with non-insulin-dependent diabetes mellitus (NIDDM) (n = 10) was compared to that from age-matched control subjects (n = 10). Platelet extract from patients with NIDDM stimulated cultured aortic endothelial cells to produce greater amounts of PGI2 than did that from control subjects. These data suggest that the increased PSA of platelets isolated from diabetic patients may contribute to the abnormal interaction between platelets and the vascular wall in diabetic patients.