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Notes: Abstract A transcriptional fusion of a synthetic FNR-dependent promoter derived from Escherichia coli has been introduced into Paracoccus denitrificans on a broad-host-range plasmid. The patterns of expression of β-galactosidase from this fusion and from a control which is not regulated by FNR have been studied. The results indicate that P. denitrificans expresses a transcriptional regulator which has a very similar DNA-binding specificity to FNR, and responds to a similar physiological signal.

Notes: A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the napA gene encoding the periplasmic nitrate reductase. This approach was used to amplify fragments of the napA gene from 10 Pseudomonas species and one Moraxella sp., previously shown to be able to express the periplasmic nitrate reductase activity, from Rhodobacter capsulatus and from community DNA extracted from a fresh-water sediment. Amino acid sequences encoded by the napA fragments were compared to one another and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocol is specific for its intended target. The napA sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. All tested Gram-negative strains capable of aerobic nitrate respiration were found to have periplasmic nitrate reductase genes. However, some strains which have and express the genes are incapable of aerobic nitrate respiration. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.

Notes: A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the narG gene encoding the membrane-bound nitrate reductase. This approach was used to amplify fragments of the narG gene from five Pseudomonas species previously shown to be able to express the membrane-bound nitrate reductase and from community DNA extracted from a freshwater sediment. Amino acid sequences encoded by the narG fragments were compared to one another, and to the corresponding regions of related enzymes. This comparison indicates that the amplification protocols are specific for their intended targets. Sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community. The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.

Notes: Abstract The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Notes: Abstract A nitrate reductase activity has been identified in periplasmic extracts of Paracoccus denitrificans. The enzyme is relatively insensitive to azide and does not reduce chlorate, features which distinguish it from the well-characterised membrane-associated nitrate reductase. The specific activity of the enzyme was higher in intact cells grown with butyrate rather than succinate as the sole source of carbon.

Notes: Abstract Bacteria which can grow in different environments have developed regulatory systems which allow them to exploit specific habitats to their best advantage. In the facultative anaerobe Escherichia coli two transcriptional regulators controlling independent networks of oxygen-regulated gene expression have been identified. One is a two-component sensor-regulator system (ArcB-A), which represses a wide variety of aerobic enzymes under anaerobic conditions. The other is FNR, the transcriptional regulator which is essential for expressing anaerobic respiratory processes.The purpose of this review is to summarize what is known about FNR. The fnr gene was initially defined by the isolation of some pleiotropic mutants which characteristically lacked the ability to use fumarate and nitrate as reducible substrates for supporting anaerobic growth and several other anaerobic respiratory functions. Its role as a transcripitonal regulator emerged from genetic and molecular studies in which its homology with CRP (the cyclic AMP receptor protein which mediates catabolite repression) was established and has since been particularly important in identifying the structural basis of its regulatory specificities. FNR is a member of a growing family of CRP-related regulatory proteins which have a DNA-binding domain based on the helix-turn-helix structural motif, and a characteristic β-roll that is involved in nucleotide-binding in CRP.The FNR protein has been isolated in a monomeric form (Mr 30 000) which exhibits a high but as yet non-specific affinity for DNA. Nevertheless, the DNA-recognition site and important residues conferring the functional specificity of FNR have been defined by site-directed mutagenesis. A consensus for the sequences that are recognized by FNR in the promoter regions of FNR-regulated genes, has likewise been identified. The basic features of genes and operons regulated by FNR are reviewed, and examples in which FNR functions negatively as an anaerobic repressor as well as positively as an anaerobic activator, are included. Less is known about the way in which FNR senses anoxia and is thereby transformed into its ‘active’s form, but it seems likely thatIt is clear that oxygen functions as a regulatory signal controlling several important aspects of mitcrobial physiology, and further studies should reveal the molecular basis of the mechanism by which changes in oxygen tension are sensed. The recent identification of FNR homologues in diverse microorganisms points to the widespread importance of this family of regulatory proteins. Moreover, the function of these proteins is not limited to the regulation of anaerobic respiration but includes roles in the regulation of nitrogen fixation and haemolysin biosynthesis. The ability to over-ride these regulatory mechanisms may have useful biotechnological applications, and it could also be important in controlling pathogenesis. It is anticipated that further studies will provide insights into the way in which these regulatory proteins with common evolutionary ancestors have diverged to regulate disparate metabolic processes.

Notes: Summary A total of 49 patients with advanced, previously untreated non-small-cell lung cancer (NSCLC) were treated with a new antifolate, Edatrexate (10-ethyl-10-deaza-aminopterin; 10-EdAM). Patients received 80 mg/m2 weekly for 12 weeks, and responders received a further 6 cycles at 2-week intervals. Dose reductions were carried out for haematological toxicity and mucositis. Response was assessed prior to each treatment according to WHO criteria. Among the 45 evaluable patients, 6 [13.3%; 95% confidence interval (CI), 6%–26%] achieved a partial response (PR) and 9 (20%; 95% CI, 11%–34%) showed a minor response (MR; 25%–50% reductions in the sum of 2 perpendicular tumour diameters). In those receiving four or more cycles of treatment, the PR and MR rates were 17.6% and 26.4%, respectively. The resultant toxicity mainly constituted skin rash, mucositis and myelosuppression. Edatrexate is active against NSCLC and produces toxicity profile similar to that of methotrexate.

Notes: Summary Chemotherapy with mitomycin C, ifosfamide and cisplatin (MIC) is reported to produce responses of 56% and 69% in inoperable non-small-cell lung cancer (NSCLC) [1,2]. We evaluated the regimen in 45 similar patients who received up to six courses of 6 mg/m2 mitomycin C, 3 g/m2 ifosfamide, and 50 mg/m2 cisplatin every 3 weeks. In all, 18 patients had limited disease (LD) and 27 had extensive disease (ED). A total of 18 patients responded (40%), 9/18 with LD and 9/27 with ED; there were 4 complete responders. The median duration of response was 25 weeks, and median survival was 32 weeks (range, 2–96 weeks). Toxicity was moderate. Nausea and vomiting were controlled with i.v. dexamethasone and high-dose metoclopramide. Other toxicities included myelosuppression and alopecia. This study confirms that MIC is one of the most active regimens for treatment of NSCLC, with acceptable toxicity.

Notes: Summary Twenty-five patients with previously untreated small cell carcinoma of the bronchus have been treated with cyclophosphamide 160–200 mg/kg and subsequent radiotherapy to the primary site. Eighty-four percent of patients responded to the single cycle of chemotherapy, with 56% attaining a complete response. Median duration of remission was 43 weeks and median survival 69 weeks. 2-Mercaptoethane sulphonate was given to prevent urothelial toxicity. Autologous bone marrow transplantation was used to mitigate bone marrow depression but sequential delay in reinfusing cryopreserved bone marrow did not alter the period of cytopenia. Other toxicities were mild. The procedure proved safe and manageable. High-dose chemotherapy may prove to be useful in the initial management of this tumour.

Notes: [Auszug] Nitric oxide (NO), synthesized in eukaryotes by the NO synthases, has multiple roles in signalling pathways and in protection against pathogens. Pathogenic microorganisms have apparently evolved defence mechanisms that counteract the effects of NO and related reactive nitrogen species. ...