Glutamine synthetase from Acetobacter diazotrophicus: properties and regulation.

Glutamine synthetase from Acetobacter diazotrophicus, an endophyte originally isolated from sugarcane, was studied as a step in the identification of mechanisms underlying the role of A. diazotrophicus as a major supplier of fixed nitrogen to its host plant. The enzyme was purified and partially characterized. It was also shown that the enzyme is regulated by adenylylation in response to the nitrogen source. Interestingly, there is no upregulation of the synthesis of the enzyme under diazotrophic conditions, which is in contrast to the situation in enterics, e.g. Klebsiella pneumoniae.

Nitrogen fertilization effects on stream water cadmium concentration.

High transition metal concentrations were previously unexpectedly observed in soil water extracted by suction lysimeters following forest N fertilization. This observation called for additional measurements to investigate if the finding is a general phenomenon and, if so, whether stream water concentrations of transition metals could increase as a result of N fertilization. The measured levels of Cd in the preliminary findings were well above health limits for drinking water. Hence, the problem could be of major concern. Here we report on soil water and stream water concentrations at two partly fertilized watersheds. All sites were situated in the central part of Sweden. The N application (150 kg N ha(-1) in the form of calcium ammonium nitrate) resulted in increased concentrations of nitrate, and a pulse of acidity through the soil profile, which increased the solubility of transition metals (mainly Cd and Zn) and Al. Stream water concentrations of transition metals, on the other hand, were not affected during the studied period by the increased solubility of transition metals in the soil. The data imply that the solubilized transition metals probably insolubilize further down the soil profile, and that there is no risk from forest N fertilization (at normal soil pH levels) of transition metal levels increasing in nearby surface waters. To our knowledge, this is the first time this side effect of N fertilization has been considered.

Episodic heavy drinking in four Nordic countries: a comparative survey.

AIMS: The purpose of this study was to compare the phenomenon of episodic heavy drinking (binge drinking) and its different indicators in the Nordic countries. DESIGN: A comparative survey of four Nordic countries. SETTING: Telephone interviews in Denmark, 1997; Finland, 1996; Norway, 1996; and Sweden 1996-97. PARTICIPANTS: Random samples of men and women aged 19-71 years. MEASUREMENTS: Episodic heavy drinking was measured by the frequency of subjective intoxication, of drinking six or more drinks at a time (6+), and of negative consequences (mainly hangover symptoms). Additionally, annual consumption and measures of intake per occasion were used. FINDINGS: Annual consumption, overall frequency of drinking and frequency of drinking 6+ were highest in Denmark and lowest in Norway. Frequency of subjectively defined intoxication was highest in Finland. There it was clearly higher than the frequency of drinking 6+, whereas in Denmark the contrary was observed. Finnish and Norwegian men and Danish women reported the largest quantities drunk per occasion. Results on 6+ frequency and the prevalence of negative consequences, with annual consumption held constant, suggest that Danes have the least concentrated drinking pattern. With annual consumption held constant, Norwegians report as high a frequency of intoxication, as do Finns. CONCLUSIONS: The relations between subjective and more objective measures of episodic heavy drinking vary considerably between the Nordic countries. The results suggest that the definition, acceptability and experience of intoxication vary even when a set of relatively homogeneous countries are compared.

Unrecorded alcohol consumption: its economics and its effects on alcohol control in the Nordic countries.

The starting point of this paper is the fact that no country has complete records of alcohol consumption. In addition to being a matter or statistical accuracy, unrecorded alcohol also plays an important role in alcohol policy discussions. Furthermore, its quantity is bound to basic economic laws. These latter two aspects are the main interest in this paper, which discusses, first, what is really meant by unrecorded alcohol consumption and what kind of categories are included in it. The next task is to discuss the economics of different categories of unrecorded alcohol and the mechanisms which lead to increases or decreases in them. The examples in this part of the paper come from the Nordic countries. Arguments about increased smuggling and illegal distilling have always been used against alcohol policy restrictions in the Nordic countries. Recently the level of travellers' alcohol imports and border trade have also been used for the same purpose. In the European Union the task to harmonize alcohol excise taxes is partly given to increased travellers' duty-free allowances and market forces. This policy has already led to reductions in alcohol taxation both in Denmark and Sweden.

Purification of P(II) and P(II)-UMP and in vitro studies of regulation of glutamine synthetase in Rhodospirillum rubrum.

The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.

Expression of P(II) and glutamine synthetase is regulated by P(II), the ntrBC products, and processing of the glnBA mRNA in Rhodospirillum rubrum.

We have studied the transcription of the glnB and glnA genes in Rhodospirillum rubrum with firefly luciferase as a reporter enzyme. Under NH(4)(+) and N(2) conditions, glnBA was cotranscribed from a weak and a strong promoter. In nitrogen-fixing cultures, activity of the latter was highly enhanced by NtrC, but transcription from both promoters occurred under both conditions. There is no promoter controlling transcription of glnA alone, supporting our proposal that the glnA mRNA is produced by processing.

Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients.

BACKGROUND: DNA vaccination is known to generate immune responses against HIV-1 in animal models. We aimed to assess the efficacy of DNA vaccination in induction of immune responses in HIV-1-infected human beings. METHODS: Nine symptom-free HIV-1-infected patients were immunised with DNA constructs encoding the nef, rev, or tat regulatory genes of HIV-1. The patients were selected for having no or low antibody reactivities to these antigens. HIV-1-specific cytotoxic T-lymphocytes (CTLs), precursor frequencies, and antigen-specific proliferative responses were measured before, during, and after three immunisations over 6 months. FINDINGS: Cellular immune reactivities against the HIV-1 regulatory proteins were absent or low before DNA immunisation. DNA vaccination induced detectable memory cells in all patients and specific cytotoxicity in eight patients. CTLs were MHC-class-I restricted and mainly of CD8+ origin. In three patients the cellular activity was transient, decreasing after an initial response. INTERPRETATION: DNA immunisation with HIV-1 genes can induce specific cellular responses in human beings with no apparent side-effects. It is theoretically possible that HIV-1-specific cytotoxic responses to regulatory proteins could lead to infected cells being eliminated before they have released new viral particles. However, it is possible that the patients we selected responded less than would non-selected or non-infected individuals. The small number of patients presented here does not allow generalisation of our findings.

Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP.

Dinitrogenase reductase-activating glycohydrolase (DRAG), involved in the regulation of nitrogenase activity in Rhodospirillum rubrum, is associated with chromatophore membranes in cell extracts. We show that DRAG can be specifically released by treatment with MgGDP; other nucleotides studied had no effect. The DRAG activity released corresponds to the release of DRAG protein.

Uridylylation of the P(II) protein in the photosynthetic bacterium Rhodospirillum rubrum.

The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.

The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum.

The role of NAD+ in the metabolic regulation of nitrogenase, the 'switch-off' effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrum devoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ 'switch-off' is an effect of the same regulatory system as ammonium 'switch-off'. We also show that oxaloacetate and alpha-ketoglutarate function as 'switch-off' effectors. On the other hand beta-hydroxybutyrate has the opposite effect by shortening the 'switch-off' period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in 'switch-off' was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating 'switch-off'.

HIV-1 epitopes exposed by hybrid hepatitis B core particles affect proliferation of peripheral blood mononuclear cells from HIV-1 positive donors.

Hepatitis B virus core protein (HBc) in particulate form is a potent immunogen for the design of vaccines with a broad T-cell reactivity. Hybrid HBc proteins with N-terminal insertions of human immunodeficiency virus (HIV-1) B- and T-cell epitopes from gp41 and p34 pol, respectively, were constructed HBc hybrids formed particles with HIV-1 epitopes exposed on the surface. The proliferative response of peripheral blood mononuclear cells (PBMC) from HIV-1 infected donors to the hybrids was studied in vitro and compared to that of synthetic peptides representing the same HIV-1 sequences. The epitope from p34 pol induced PBMC proliferation both when inserted into HBc and as a peptide. The epitope from gp41, when inserted into HBc, and to a lesser extent the relevant peptide caused a decreased reactivity. The inhibitory effect of the HBc hybrid carrying the gp41-epitope was pronounced even in the HIV-1 infected donors with proliferative responses to HBc. The similarity between the mode of action of the peptides and hybrid HBcs implies either correct processing of the latter or T-cell recognition of HIV-1 epitopes in the intact hybrid HBc particles.

Identification and sequence of a nifJ-like gene in Rhodospirillum rubrum: partial characterization of a mutant unaffected in nitrogen fixation.

A nifJ-like gene was identified in the photosynthetic purple non-sulphur bacterium Rhodospirillum rubrum. A DNA segment hybridizing to Klebsiella pneumoniae nifJ was isolated, the gene was inactivated, and a mutant strain, SNJ-1, was constructed by allele replacement. The mutation was confirmed by DNA sequencing. Northern blotting and by the lack of pyruvate oxidoreductase activity. This is the first report of a nifJ-like gene in photosynthetic bacteria. Unexpectedly, SNJ-1 was capable of nitrogen fixation, and growth was similar to the wild-type strain under all conditions investigated. Therefore, this is also the first demonstration that a nifJ homologue, when present, is not essential for nitrogen fixation in a diazotroph. The nifJ-like gene was sequenced and found to have considerable similarity to published nifJ gene sequences from other organisms. By primer extension, the initiation site for transcription was located, and a typical sigma 54 promoter sequence was identified.

Potential consequences from possible changes to Nordic retail alcohol monopolies resulting from European Union membership.

This paper projects the consequences of modifying or eliminating the current national alcohol retail monopolies in Sweden, Norway and Finland as a possible result of those countries' membership in the European Union (EU). First, the authors project absolute alcohol consumption in each country based on different possible changes in alcohol price and availability. Then they predict the future levels of alcohol-related problems likely to result from increased per capita alcohol consumption (Sweden and Norway only). All of the scenarios examined in this paper are expected to lead to increases in per capita alcohol consumption. The smallest increase in consumption would result from a partial elimination of the current monopoly and a modest reduction in alcohol prices. In that case, projected per capita consumption in Sweden for inhabitants 15 years and older would rise from 6.3 to 9.3 litres; in Norway, from 4.7 to 6.7 litres; and in Finland, from 8.4 to 11.1 litres. The greatest projected increase in consumption would result from a complete elimination of the state monopolies such that all beer, wine and spirits were sold in food shops, grocery stores and gasoline stations, along with a substantial drop in alcohol prices as a result of private competition within each country and increased cross-border alcohol purchases. That scenario would result in projected per capita consumption of 12.7 litres in Sweden, 11.1 litres in Norway and 13.7 litres in Finland. The authors project that a 1-litre increase in consumption would result in a 9.5% increase in total alcohol-related mortality in Sweden and a 9.7% increase in Norway. Further, alcohol-related assaults would increase by 9% in Sweden and 9.6% in Norway. A 5-litre increase in consumption would result in a 62% increase in alcohol-related mortality in Sweden and a 60% increase in Norway, and a 57% increase in alcohol-involved assaults in both countries.

Changes in the NAD(P)H concentration caused by addition of nitrogenase 'switch-off' effectors in Rhodospirillum rubrum G-9, as measured by fluorescence.

The effect of nitrogenase 'switch-off' effectors on the concentration of NAD(P)H in Rhodospirillum rubrum G-9 was investigated by fluorescence. A rapid decrease in fluorescence was observed when cells, either N2-grown or nitrogen-starved, were subjected to the effectors, but not when sodium chloride or Tris buffer was added. No effects on the fluorescence were observed in non-nitrogen fixing cultures except when NAD+ was added. The results strongly indicate that the redox state of the pyridine nucleotide pool affects the control of the regulation of nitrogenase activity in R. rubrum.

Immunological and virological interactions in patients receiving passive immunotherapy with HIV-1 neutralizing monoclonal antibodies.

Mouse monoclonal antibodies with high human immunodeficiency virus type 1 (HIV-1) neutralizing titers were used for passive immunotherapy of eleven late-state HIV-infected patients. In five patients the serum level of the core protein p24 decreased, while in five cases it remained unchanged. The level of viral RNA in plasma as measured by quantitative polymerase chain reaction (PCR) decreased in four cases, was stable in another four, and increased in three cases. An anti-mouse (HAMA) response developed in eight patients and anti-idiotypic antibodies appeared in six. Immune complexes that formed in patient sera during the treatment were shown to contain mostly envelope glycoprotein gp120 which decreased in nine of the eleven treated patients toward the end of treatment. Antibodies inhibiting gp120 binding to CD4 became detectable or increased in six patients during immunotherapy. Serology of the HIV-1 V3 region was studied for both the HIV-1 IIIB and MN strains with no or very small changes in titer or avidity after treatment. No change in neutralizing titers to strain HTLVIIIB was observed in serum samples collected before and after treatment was terminated. In nine of the eleven patients stimulation of the T lymphocytes to proliferate in vitro when activated by phytohemagglutinin (PHA) was shown to be increased compared to before treatment. Increased T-cell proliferation was also noted with several antigens such as HIV-1 recombinant antigens, cytomegalovirus (CMV), tetanus toxoid (TT), and purified protein derivate of mycobacterium tuberculosis (PPD). These findings indicate a decreased total gp120 content in serum, permitting better T-cell activation.

Benchmarking: finding ways to improve.

BACKGROUND: This article provides health care organizations with a benchmarking methodology to use in comparing practices and processes to identify and actualize opportunities for improvement. The 15-step model can be used to guide internal and external benchmarking activities in a variety of health care settings. EXAMPLES: Two internal benchmarking case studies are illustrated: (1) one hospital used the Baxter model to evaluate its laparoscopic cholecystectomy services, and (2) another prepared for expansion of cardiac catheterization services. CONCLUSIONS: Two key factors of a successful benchmarking initiative are highlighted: management commitment and involvement, and the need to understand internal practices and processes before benchmarking.

Randomized response estimates for the purchase of smuggled liquor in Norway.

The randomized response technique is applied on a voluntary basis for estimating the purchase of smuggled liquor in Norway. Of a total sample of 2330 respondents, aged 15 and over, about one half chose the protective option of the RR-technique. The other half responded directly to the supposed sensitive questions. The estimates from the two groups were weighted together to give population estimates for the percentage of buyers, and the quantities bought, of smuggled liquor. The resulting estimates were found to be considerably higher than estimates from a contemporary survey based on direct response only.

Improved cell-mediated immune responses in HIV-1-infected asymptomatic individuals after immunization with envelope glycoprotein gp160.

Strong specific T-cell responses to human immunodeficiency virus type 1 (HIV-1) gp160 were induced by immunization with recombinant gp160 (rgp160). It was given as postinfection vaccination to 40 asymptomatic HIV-1 seropositive patients. The participants received 6 doses of 160 micrograms rgp160 administered intramuscularly at 0, 1, 4, 8, 17, and 26 weeks and were monitored for 1 year. Lymphocyte proliferation was performed by cultivating lymphoid cells in vitro with specific antigens and mitogens. After immunization with gp160, specific T-cell proliferative responses were induced in all 40 patients. One week after the sixth immunization at day 180, a substantially increased response was detected in 98% of the patients, with a mean stimulation index value of 195. Furthermore, proliferative responses were also identified, after immunization, against native gp120 and against a peptide representing the V3 region of gp120. In addition to the HIV-specific T-cell responses, increased reactivity to several other non-HIV antigens, including tetanus toxoid, influenza, measles, and cytomegalovirus, were seen after gp160 vaccination. The responses to CMV and measles were interpreted to represent an improved recall antigen response. Such recall antigen responses were few in matched HIV-infected controls immunized with influenza virus only. All patients initially and repeatedly showed a normal capacity of total T-cell activation, evaluated by the mitogen phytohemagglutinin (PHA). The trend in CD4 counts improved in 30 of 40 patients during the year of follow-up. The frequency of increases of proliferative responses to antigens was associated with a better CD4 trend. Addition of zidovudine for 2 weeks after each immunization had no beneficial effects nor did it prevent induction of immune responses. All patients tolerated the immunizations well, and no systemic adverse effects were noted. This is a phase I trial, and no definitive conclusions regarding clinical efficacy can be reached.

Visiting public drinking places in Oslo: an application of the theory of planned behaviour.

This study was undertaken to describe and obtain better insight into pub-going in the Norwegian capital applying the theory of planned behaviour (TPB). The sample comprised 1053 persons aged between 16 and 71 years who visited public drinking places in Oslo. Two thirds of the sample were men. Data were collected by means of anonymous self-administered questionnaires. The majority of the respondents reported having visited a public drinking place at least once a week during the past 12 months. In the applied model, intention to visit a public drinking place during the next 14 days is considered to be a joint function of the attitude towards pub-going (Aact), subjective norms (SN), and perceived control over the behaviour (PBC). The goodness of fit (R2) of the model was 22%. The relative importance of the model's components were in descending order (beta): PBC, Aact and SN. Previous behaviour was included as a predictor in the TPB, and had a stronger effect upon intention than the model's original components. In conclusion, the TPB provided an adequate understanding of why pub and cafe guests frequent public drinking places. However, it is possible that the model over-simplifies the processes connected to pub-going and fails to describe the dynamics of the relationship between the individual and the environment.