Transcriptional control, translation and function of the products of the five open reading frames of the Escherichia coli nir operon

Five open reading frames designated nirB, nirD, nirE, nirC and cysG have been identified from the DNA sequence of the Escherichia coli nir operon. Complementation experiments established that the NirB, NirD and CysG polypeptides are essential and sufficient for NADH-dependent nitrite reductase activity (EC 1.6.6.4). A series of plasmids has been constructed in which each of the open reading frames has been fused in-phase with the beta-galactosidase gene, lacZ. Rates of beta-galactosidase synthesis during growth in different media revealed that nirB, -D, -E and -C are transcribed from the FNR-dependent promoter, p-nirB, located just upstream of the nirB gene: expression is co-ordinately repressed by oxygen and induced during anaerobic growth. Although the nirB, -D and -C open reading frames are translated into protein, no translation of nirE mRNA was detected. The cysG gene product is expressed from both p-nirB and a second, FNR-independent promoter, p-cysG, located within the nirC gene. No NADH-dependent nitrite reductase activity was detected in extracts from bacteria lacking either NirB or NirD, but a mixture of the two was as active as an extract from wild-type bacteria. Reconstitution of enzyme activity in vitro required stoichiometric quantities of NirB and NirD and was rapid and independent of the temperature during mixing. NirD remained associated with NirB during the initial stages of purification of the active enzyme, suggesting that NirD is a second structural subunit of the enzyme.     

Development and characterization of a human cell line from an ovarian mixed mullerian tumor (carcinosarcoma)

Summary A cell line derived from a human ovarian carcinosarcoma was established in tissue culture and in nude mice. Two sublines, LDF and HDF, separated by discontinuous density centrifugation were also established from the parent line JoN. The cloning efficiency of the JoN line was 21%. Morphologic features of adenocarcinoma cells characteristic of the parent JoN cells were retained in the sublines and clones; all lines showed the same karyotype and DNA content (pseudodiploid and pseudotetraploid). Keratin, as demonstrated immunohistochemically, was strongly expressed in the parent line JoN and the xenograft tumor, but not at all in the LDF sublines and only moderately in the HDF sublines. Vimentin, however, was expressed in neither the parent line JoN nor the xenograft tumor, but was present in both sublines. Transglutaminase and plasminogen activator activity was high in the parent line JoN. Neither, sublines nor clones showed the same high enzyme activity as the parent line. It is concluded that this human tumor line JoN is comprised of epithelial cells, capable of multidirectional differentiation.     

Competitive interactions between corticosterone and 11-dehydrocorticosterone in binding to receptors in fetal mouse tissues

The binding of ³H-corticosterone and ³H-11-dehydrocorticosterone to receptors in cytosol and nucleus was examined in fetal mouse brain and placenta using Sephadex gel filtration or charcoal to separate bound and unbound steroid. In the cytosol, competitive displacement of each steroid by the other was observed. The binding was unaffected by RNase, DNase, dithiothreitol or N-ethyl maleimide but was diminished by Pronase. Nuclei were isolated by hypotonic shock using dilute MgCl₂ and the steroid receptor-complexes of both steroids were obtained from the nuclear sap. Receptor-complexes of both steroids were observed in brain and placental tissues. Competitive displacement of each steroid by the other was also observed in nuclear binding. Both 11-dehydrocorticosterone and 11-deoxycorticosterone bound to a chromatin fraction as did the hormone corticosterone. Identity of the steroids was established by using chromatography and co-crystallization techniques. This work raises the possibility that in the fetal mouse, 11-dehydrocorticosterone, previously considered biologically inactive and an abundant metabolite in fetal mouse tissues, may in fact play a more positive role in regulation.     

Some comparisons of cellulases from two different strains of aspergillus fumigatus

Michaelis-Menten kinetics for exoglucanase, endoglucanase and β-glucosidase activities from two different strains of Aspergillus fumigatus were compared in the absence and the presence of ammonium ions. Inhibitory effects, evident in only one strain, were quantified, suggesting non-competitive inhibition for endoglucanase and β-glucosidase, but competitive inhibition of exocellulase. Possible reasons are discussed.     

PERIZONIUM AND INITIAL VALVE FORMATION IN THE DIATOM NAVICULA CUSPIDATA (BACILLARIOPHYCEAE)1

This paper describes the perizonium and initial valve formation in Navicula cuspidata Kütz., based on light microscope (LM) and scanning electron microscope (SEM) observations. The perizonium consists of concentric over-lapping bands, laid down sequentially at the tips of the expanding biconical auxospore during its elongation. The central perizonial band has fimbriate edges and is considerably more rigid than the more distal bands. During auxospore elongation and the band secretion, the chloroplasts continuously oscillate between the two ends of the cell; this oscillation ceases once the elongation is complete. The initial valves, formed within the perizonium, are molded into the basically biconical shape of the perizonium except for a central flattening of each valve face. In contrast to the raphes in gametangial and vegetative valves which are surrounded by a smooth axial area, the raphes in initial valves lie within a raised ridge running along the apical axis of the valve. The regular pattern of apically oriented ridges on the outer surface of vegetative valves is also lacking on initial valves. Comparison of pore–pore spacing within striae of gametangial valves, initial values and post-initial valves (first division and vegetative cells) reveals that the pore–pore distance within striae is conserved at all sexual stages. However, the distance between striae is considerably larger in initial valves than in gametangial and post-initial valves. Vegetative interstriae spacing as well as the planar morphology of the valve face is regained at the first division of the initial cell. This suggests that the spacing between striae is dependent on the sexual stage of the cell during valve formation (i.e. not directly dependent on the cell size) and can be altered independently of the pore–pore spacing.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.     

The effects of dogfish MSH''s and of corticotrophin-like intermediate lobe peptides (CLIP''s) on avoidance behavior in rats

Recently purified melanocyte-stimulating hormones (MSH's) from dogfish pituitary tissue were tested on extinction of a conditioned avoidance response (CAR). Corticotrophin like intermediate lobe peptides (CLIP's) from dogfish and porcine origin were tested for an effect on avoidance extinction as well. All peptides appeared to delay extinction of the CAR.The results suggest that the pituitary contains various peptides which influence adaptive behavior. The observation that MSH is more potent in delaying extinction of the CAR then CLIP leads to the conclusion that the behavioral active sequence of the ACTH molecules is located in the N terminal part rather than in the C terminal part of the polypetide.     

Evaluation of the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age: The DESiGN cluster randomised trial

BackgroundAntenatal detection and management of small for gestational age (SGA) is a strategy to reduce stillbirth. Large observational studies provide conflicting results on the effect of the Growth Assessment Protocol (GAP) in relation to detection of SGA and reduction of stillbirth; to the best of our knowledge, there are no reported randomised control trials. Our aim was to determine if GAP improves antenatal detection of SGA compared to standard care.Methods and findingsThis was a pragmatic, superiority, 2-arm, parallel group, open, cluster randomised control trial. Maternity units in England were eligible to participate in the study, except if they had already implemented GAP. All women who gave birth in participating clusters (maternity units) during the year prior to randomisation and during the trial (November 2016 to February 2019) were included. Multiple pregnancies, fetal abnormalities or births before 24⁺¹ weeks were excluded. Clusters were randomised to immediate implementation of GAP, an antenatal care package aimed at improving detection of SGA as a means to reduce the rate of stillbirth, or to standard care. Randomisation by random permutation was stratified by time of study inclusion and cluster size. Data were obtained from hospital electronic records for 12 months prerandomisation, the washout period (interval between randomisation and data collection of outcomes), and the outcome period (last 6 months of the study). The primary outcome was ultrasound detection of SGA (estimated fetal weight <10th centile using customised centiles (intervention) or Hadlock centiles (standard care)) confirmed at birth (birthweight <10th centile by both customised and population centiles). Secondary outcomes were maternal and neonatal outcomes, including induction of labour, gestational age at delivery, mode of birth, neonatal morbidity, and stillbirth/perinatal mortality. A 2-stage cluster–summary statistical approach calculated the absolute difference (intervention minus standard care arm) adjusted using the prerandomisation estimate, maternal age, ethnicity, parity, and randomisation strata. Intervention arm clusters that made no attempt to implement GAP were excluded in modified intention to treat (mITT) analysis; full ITT was also reported. Process evaluation assessed implementation fidelity, reach, dose, acceptability, and feasibility. Seven clusters were randomised to GAP and 6 to standard care. Following exclusions, there were 11,096 births exposed to the intervention (5 clusters) and 13,810 exposed to standard care (6 clusters) during the outcome period (mITT analysis). Age, height, and weight were broadly similar between arms, but there were fewer women: of white ethnicity (56.2% versus 62.7%), and in the least deprived quintile of the Index of Multiple Deprivation (7.5% versus 16.5%) in the intervention arm during the outcome period. Antenatal detection of SGA was 25.9% in the intervention and 27.7% in the standard care arm (adjusted difference 2.2%, 95% confidence interval (CI) −6.4% to 10.7%; p = 0.62). Findings were consistent in full ITT analysis. Fidelity and dose of GAP implementation were variable, while a high proportion (88.7%) of women were reached. Use of routinely collected data is both a strength (cost-efficient) and a limitation (occurrence of missing data); the modest number of clusters limits our ability to study small effect sizes.ConclusionsIn this study, we observed no effect of GAP on antenatal detection of SGA compared to standard care. Given variable implementation observed, future studies should incorporate standardised implementation outcomes such as those reported here to determine generalisability of our findings.Trial registrationThis trial is registered with the ISRCTN registry, ISRCTN67698474.

Matias C Vieira and colleagues evaluate the Growth Assessment Protocol (GAP) for antenatal detection of small for gestational age in the DESiGN cluster randomised trial.