Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Background  

Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.     

A model of dengue fever

Background  

Dengue is a disease which is now endemic in more than 100 countries of Africa, America, Asia and the Western Pacific. It is transmitted to the man by mosquitoes (Aedes) and exists in two forms: Dengue Fever and Dengue Haemorrhagic Fever. The disease can be contracted by one of the four different viruses. Moreover, immunity is acquired only to the serotype contracted and a contact with a second serotype becomes more dangerous.     

Melting experiment concerning the topological structure of closed circular double-stranded DNA

A melting experiment was performed on the whole set of populations of the replicative form of ?X174 DNA, which can be obtained treating this DNA with rat liver nicking-closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double-stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double-stranded DNA: for native ?X174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpretation.     

Improved methods for the delivery of liposome-sequestered RNA into eucaryotic cells

RNA sequestered by negatively charged liposomes becomes cell-associated following interaction between eucaryotic cells and the liposomes. This paper provides evidence that cell-associated RNA is internalized by the cells. In fact, (a) when Escherichia coli and mammalian RNA are entrapped within the same liposome population and delivered into cultured cells, one can observe degradation of the procaryotic but not the eucaryotic RNA. Such an event cannot happen extracellularly. (b) Scanning electron microscopy reveals no more than 10 liposomes adhering to each cell upon liposome-cell interaction under conditions in which the RNA entrapped by 140 liposomes becomes associated with each cell. The ability of liposomes prepared by (a) the cochleate process, (b) the reverse-phase evaporation technique, and (c) the ether infusion technique, to sequester and deliver RNA into cells was investigated. Reverse-phase evaporated liposomes were most efficient in sequestering RNA (20–40%), however, all types of liposomes delivered RNA with comparable efficiency. The rate of liposome-mediated RNA delivery into mammalian cells could be substantially improved when: (a) liposome-cell interaction was carried out at pH 6.5 (twofold increase over pH 7.5), (b) a basic protein (methylated albumin) was present (two- to threefold increase), (c) liposome-cell cultures were treated with polyethylene glycol 6000 (four- to eight-fold increase), and (d) DEAE-dextran was added during interaction of liposomes with cell monolayers (four- to eight-fold increase).     

Morning-to-evening differences in oxygen uptake kinetics in short-duration cycling exercise

This study analyzed diurnal variations in oxygen (O(2)) uptake kinetics and efficiency during a moderate cycle ergometer exercise. Fourteen physically active diurnally active male subjects (age 23+/-5 yrs) not specifically trained at cycling first completed a test to determine their ventilatory threshold (T(vent)) and maximal oxygen consumption (VO(2max)); one week later, they completed four bouts of testing in the morning and evening in a random order, each separated by at least 24 h. For each period of the day (07:00-08:30 h and 19:00-20:30 h), subjects performed two bouts. Each bout was composed of a 5 min cycling exercise at 45 W, followed after 5 min rest by a 10 min cycling exercise at 80% of the power output associated with T(vent). Gas exchanges were analyzed breath-by-breath and fitted using a mono-exponential function. During moderate exercise, the time constant and amplitude of VO(2) kinetics were significantly higher in the morning compared to the evening. The net efficiency increased from the morning to evening (17.3+/-4 vs. 20.5+/-2%; p<0.05), and the variability of cycling cadence was greater during the morning than evening (+34%; p<0.05). These findings suggest that VO(2) responses are affected by the time of day and could be related to variability in muscle activity pattern.     

Radiation biology of mosquitoes

There is currently renewed interest in assessing the feasibility of the sterile insect technique (SIT) to control African malaria vectors in designated areas. The SIT relies on the sterilization of males before mass release, with sterilization currently being achieved through the use of ionizing radiation. This paper reviews previous work on radiation sterilization of Anopheles mosquitoes. In general, the pupal stage was irradiated due to ease of handling compared to the adult stage. The dose-response curve between the induced sterility and log (dose) was shown to be sigmoid, and there was a marked species difference in radiation sensitivity. Mating competitiveness studies have generally been performed under laboratory conditions. The competitiveness of males irradiated at high doses was relatively poor, but with increasing ratios of sterile males, egg hatch could be lowered effectively. Males irradiated as pupae had a lower competitiveness compared to males irradiated as adults, but the use of partially-sterilizing doses has not been studied extensively. Methods to reduce somatic damage during the irradiation process as well as the use of other agents or techniques to induce sterility are discussed. It is concluded that the optimal radiation dose chosen for insects that are to be released during an SIT programme should ensure a balance between induced sterility of males and their field competitiveness, with competitiveness being determined under (semi-) field conditions. Self-contained ⁶⁰Co research irradiators remain the most practical irradiators but these are likely to be replaced in the future by a new generation of high output X ray irradiators.     

Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Enhancement of oxidative DNA degradation by histidine: the role of stereochemical parameters.

The nicking of supercoiled DNA by H2O2 and ferrous iron has been studied in a variety of environmental conditions. The replicative form of phage fd DNA (fd RF DNA) was used for investigating the phenomenon. The rate of nicking was measured in 10 mM NaCl. The addition of 1 mM Tris-HCl buffer (pH 7.5) slowed down the rate of nicking, the addition of 0.1 mM histidine enhanced it. The simultaneous presence of 1 mM Tris-HCl buffer and of 0.1 mM histidine further enhanced the rate of nicking of fd RF DNA. Increasing the concentration of NaCl dramatically reduced the rate of the reaction. The degradation of fd RF DNA was determined as a function of the concentration of histidine (0-5 mM): the rate increases with concentration, reaches a maximum and then decreases. In the presence of histidine, increasing the concentration of Tris leads to a similar phenomenon. In the absence of histidine, Tris always quenches the degradation of DNA. Electron spin resonance measurements failed to detect an enhancement of the signal characteristic for the hydroxyl radical when histidine was added to the solution containing hydrogen peroxide and ferrous iron. When the nicking of DNA is achieved via the process of auto-oxidation of ferrous iron (i.e., in the absence of added H2O2), histidine only reduces the rate of reaction in a dose-dependent manner, in the explored range of concentrations. In the presence of H2O2 and ferrous iron, histidine enhances the rate of nicking of double-stranded DNA in its supercoiled as well as in its relaxed state, but fails to modify the rate of nicking of fd DNA when it is in its vegetative, single-stranded form.     

The origin of DNA:RNA hybridization

Conclusions Besides its use in basic research, the DNA:RNA hybridization technique has helped the development of genetic engineering: it is instrumental in the isolation of specific genes that can be inserted into foreign cells, thus modifying their genetic information. Plants, animals, and microorganisms can now be altered to yield improved crops, pest-resistant plants, and a cheaper source of important proteins or drugs. The social relevance of genetic engineering received official sanction in 1980 when the U.S. Supreme Court ruled that genetically modified organisms can be patented. In this article I have tried to describe the discovery of the DNA:RNA hybridization technique as the successful outcome of years of intelligent and patient research in many laboratories, of inductive and deductive processes in the minds of many biologists. The synthesis that led to the final result and to the early development of the technique was made possible by the coming together of two brilliant scientists, Sol Spiegelman and Benjamin Hall.