The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.     

Genotoxic effects of a sub-acute low-level inhalation exposure to a mixture of carcinogenic chemicals

A study was conducted using a combined testing protocol (CTP), to determine whether short-term biological end-points, singly or in combination, are sufficiently sensitive to identify damage induced by exposure to ambient levels of industrial chemicals. A small-scale inhalation set-up which is both economical and easy to assemble was designed. Mice were exposed to 4 concentrations of a custom-blend mixture of benzene, chloroprene, epichlorohydrin and xylene in a ratio of 2:2:1:2, respectively. The concentrations for benzene, chloroprene and xylene were 0, 0.1, 1.0 and 10 ppm each. Concentrations for epichlorohydrin were half those for the other components. Groups of 22 males and 22 female mice were exposed to each concentration of the mixture for 3 and 6 weeks. Selected biological end-points including urine mutagenesis, bone marrow cell aberrations and micronuclei, spleen lymphocyte aberrations and liver enzyme induction were monitored. The spleen lymphocyte aberrations and liver enzyme induction were the most sensitive end-points. The lymphocytes showed a significant induction of chromosome aberrations from exposure for 3 weeks to all 3 concentrations of the mixtures. After 6 weeks of exposure, significant induction of aberrations was observed after exposure to low and medium concentrations but not to the high concentration. This lack of response at the high concentration after 6 weeks exposure, appeared to correlate with a significant induction of glutathione S-transferase in the liver. Since this enzyme is known to detoxify 3 of the 4 chemicals in our mixture, it may indicate a detoxification mechanism after enzyme induction. The present study indicates that the CTP is sufficiently sensitive to identify toxicological effects after exposure to ambient levels of a gas mixture.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.     

Ba2+ release from soda glass modifies single maxi K+ channel activity in patch clamp experiments.

Glasses used to fabricate patch pipettes may release components which affect ion channels (Cota, G., and C.M. Armstrong. 1988. Biophys. J. 53:107-109; Furman, R.E., and J.C. Tanaka. 1988. Biophys. J. 53:287-292; Rojas, L., and C. Zuazaga. 1988. Neurosci. Lett. 88:39-44). The gating properties of maxi K+ channels from Necturus gallbladder epithelium depend on whether borosilicate glass (BG) or blue tip hematocrit glass (SG) is used to construct the patch pipettes. The data are consistent with solubilization from SG of a component which exerts voltage-dependent, cytosolic-side specific block, closely resembling "slow block" by Ba2+ ions. Ringer's solution preincubated with SG, but not with BG, blocked inside-out maxi K+ channels when used as bathing solution. Mass spectrometry revealed that Ba2+ is released by the glass from fast and slow-release compartments (SG contains 3% wt/wt BaO), and is the only ion found in the solution at concentrations consistent with the observed channel block. Additionally, SG released O2-, Na+, Ca2+, and Mg2+, all to micromolar concentrations. These elements do not interfere with maxi K+ channels but they could in principle alter the properties of other ion channels. Thus, screening for channel-modifying substances released by the glass may be necessary for the adequate interpretation of patch-clamp results.     

Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson’s Disease

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Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate.

Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped [g]DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-DNA complexes were analyzed by electrophoresis on agarose gels and visualized by autoradiography. The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substrates and from those formed in the absence of gamma-S-ATP. Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP, and the order of preference of binding was gDNA > dsDNA > ssDNA. Since gDNA has both ds- and ssDNA components, we suggest that the role for ATP in RecF protein-DNA interactions in vivo is to confer specificity of binding to dsDNA-ssDNA junctions, which is necessary for catalyzing DNA repair and recombination.     

Palaeoclimatic conditions in the upper Pleistocene and Holocene Bhimtal-Naukuchiatal lake basin in south-central Kumaun, North India

A 52 m thick upper Pleistocene and Holocene terrestrial succession in the Bhimtal-Naukuchiatal basin, south-central Kumaun Himalaya, India was studied using chronological, palaeontological, palynological and δ¹³C measurements. The section recorded evidence for climatic changes. At least two phases of arid climate and one phase of humid climate were recognised. Preliminary palaeomagnetic studies revealed a reversal of polarity, presumably correlatable with the Mono Lake excursion. Prior to this, no reversal event in the upper Pleistocene-Holocene terrestrial sediments of Indian subcontinent is known. A fossiliferous horizon, discovered in the lower part of the section, consisted of Sorex and Mus. This is the only report of a Late Pleistocene micromammalian assemblage in the Kumaun Himalaya.     

Influence of COD/NH3–N ratio on organic removal and nitrification using a modified RBC

Synthetic wastewaters were prepared with different influent concentrations of ammonia nitrogen (NH₃–N) and COD and the treatment studies were conducted using a rotating biological contactor (RBC). If organic removal and nitrification can be simultaneously effected in one process, it will be an ideal solution to water pollution control. The RBC used in the present study was a four stage laboratory model and the discs were modified by attaching porous netlon sheets to enhance biofilm area. The COD loads (S ₀) used were about 1000 and 1500?mg/l whereas NH₃–N concentrations used were in the range of 20 to 185?mg/l. Hydraulic load (q) of 0.03?m³?.?m^-2?.?d^-1 and ammonia nitrogen loadings in the range of 0.66 to 5.5?g NH₃–N?.?m^-2?.?d^-1 were used. The RBC was operated at two different rotating speeds of 6 and 12?rpm. The results showed that the nitrification and percentage of COD removal were not affected up to the value of the COD/NH₃–N in the range from 47 to 23 at w=6?rpm and for an average influent COD of 1003?mg/l. Beyond that range only the nitrification rate decreased much whereas the percentage of COD removal was not affected. Similarly, at an influent COD load of 1557?mg/l, the nitrification and percentage COD removal were not affected for the value of the COD/NH₃–N in the range from 44 to 23 but beyond that range only the nitrification rate decreased while the percentage of COD removal was approximately constant and still high. A correlation plot between the NH₃–N removed and NH₃–N applied was presented at a rotating speed of 6?rpm and it was found that the nitrification rate of 3.93?g NH₃–N?.?m^-2?.?d^-1 was achieved at ammonia loading of 5.55?g NH₃–N?.?m^-2?.?d^-1. Also the results at w=12?rpm showed improvement of nitrification rate over those at 6?rpm.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol