Room-temperature synthesis of a new stable (N2H4)WO3 compound: a route for hydrazine trapping.

A new (N2H4)WO3 compound has been obtained by mixing WO3 and aqueous hydrazine solution at room temperature for 24 h. The reaction is catalyzed by the presence of lithium. X-ray, synchrotron and neutron diffraction techniques have shown that the material crystallizes in trigonal space group P3221 (No. 154). Chains of distorted WO4 tetrahedra extend along the a axis of the unit cell, linked by a corner-sharing oxygen atom: the N2H4 are in the voids between them. The thermal characterization shows that this new compound is stable up to 220°C, greatly beyond the boiling point of N2H4 (114°C); thus making it a promising candidate for catalysis or trapping applications.

Biofilms of As(III)-oxidising bacteria: formation and activity studies for bioremediation process development.

The formation and activity of an As(III)-oxidising biofilm in a bioreactor, using pozzolana as bacterial growth support, was studied for the purpose of optimising fixed-bed bioreactors for bioremediation. After 60 days of continuous functioning with an As(III)-contaminated effluent, the active biofilm was found to be located mainly near the inflow rather than homogeneously distributed. Biofilm development by the CAsO1 bacterial consortium and by Thiomonas arsenivorans was then studied both on polystyrene microplates and on pozzolana. Extra-cellular polymeric substances (EPS) and yeast extract were found to enhance bacteria attachment, and yeast extract also appears to increase the kinetics of biofilm formation. Analysis of proteins, sugars, lipids and uronic acids indicate that sugars were the main EPS components. The specific As(III)-oxidase activity of T. arsenivorans was higher (by ninefold) for planktonic cells than for sessile ones and was induced by As(III). All the results suggest that the biofilm structure is a physical barrier decreasing As(III) access to sessile cells and thus to As(III)-oxidase activity induction. The efficiency of fixed-bed reactors for the bioremediation of arsenic-contaminated waters can be thus optimised by controlling different factors such as temperature and EPS addition and/or synthesis to increase biofilm density and activity.

A rapid reversed phase high performance liquid chromatographic method for determination of etoposide (VP-16) in human plasma.

A rapid, simple and sensitive isocratic High Performance Liquid Chromatography (HPLC) method was developed to measure the concentration of etoposide in plasma samples with UV detection at 220 nm. The method uses a Bondapac C18 column at 60 degrees C. The mobile phase consists of Methanol: water (45:55 v/v) at a flow rate of 2.8 ml/min. Phenacetin was used as an internal standard. The plasma samples were extracted using ether with the organic layer evaporated under nitrogen. The residue was dissolved in 200 microl methanol with 20 microl injected into the HPLC column. The extraction method showed a recovery of 91.5+/-3% for etoposide. In this system, the retention time of phenacetin and etoposide were 3.3 and 4.4 min, respectively. The limit of detection of etoposide in plasma is 20 ng/ml and the limit of quantitation is 40 ng/ml. This analytical method has very good reproducibility (8.1% between-day variability at a concentration of 50 ng/ml). It is a fast, sensitive and economic method applicable for clinical and pharmacokinetic studies.

A rapid reversed phase high performance liquid chromatographic method for the determination of docetaxel (Taxotere) in human plasma using a column switching technique.

A rapid, simple and sensitive isocratic high performance liquid chromatography (HPLC) method was developed to measure the concentration of docetaxel in plasma samples with UV detection at 227 nm. The method uses a column switching technique with an Ultrasphere C18 column (75 x 4.6 mm ID, 3 mu, Altex, USA) as clean-up column and a CSC-nucleosil C8 column (150 x 4.6 mm ID, 5 mu, CSC, Montreal, Canada) as the analytical column. The mobile phase consisted of Phosphate buffer (30 mM, pH = 3)-acetonitrile (47:53, v/v) with the flow rates of 1.1 and 1.3 ml min-1 for clean-up and analytical columns, respectively. Paclitaxel was used as an internal standard. The plasma samples were extracted using a solid phase extraction method with Ammonium acetate (30 mM, pH = 5)-acetonitrile (50:50, v/v) as final eluent. The extraction method showed a recovery of 92% for docetaxel. In this system, the retention times of docetaxel and Paclitaxel were 7.2 and 8.5 min, respectively. The detection limit of docetaxel in plasma is 2.5 ng ml-1. This analytical method has a very good reproducibility (7.2% between-day variability at a concentration of 10 ng ml-1). It is applicable in clinical and pharmacokinetic studies.

The low molecular weight heparin, enoxaparin, limits infarct size at reperfusion in the dog.

OBJECTIVE: Heparin (HEP) is used in the post-thrombolytic state to prevent vessel reocclusion, thereby aiding myocardial salvage. Side effects limit its benefits, but besides anticoagulant activity HEP has diffuse actions that may be potentially beneficial to jeopardized reperfused myocardium. This study compares the effect of therapeutic doses of HEP and enoxaparin (ENOX), a low molecular weight heparin, and to streptokinase (SK), on infarct size. METHODS: The left anterior descending coronary artery was occluded in dogs for 90 min, followed by 6 h of reperfusion with a residual critical stenosis in place. Five min before reperfusion, HEP (2800 IU) was injected i.v., and perfused at 500 IU/h until sacrifice in group 2, while groups 3 and 4 received ENOX (2128 anti-Xa IU i.v.) followed by 380 anti-Xa IU/h. Group 4 was also given 500,000 IU SK over 30 min before reperfusion beginning at 55 min of occlusion (ENOX + SK), while group 5 received only SK. Controls (CON, group 1) received saline. P-selectin mediated platelet-neutrophil rosettes formation was also tested in vitro in the presence of HEP and ENOX. RESULTS: The area at risk delimited by dye perfusion was statistically similar among groups. Covariance analysis between infarct size (% of area at risk) delimited with triphenyltetrazolium and collateral flow measured with radioactive microspheres confirmed that groups given ENOX (21.6 +/- 5.5%) and ENOX + SK (24.9 +/- 3.9%) developed smaller infarcts (P < 0.05) than CON (48.1 +/- 4.5%), as opposed to HEP (32.2 +/- 3.6%) and SK (46.8 +/- 3.4%) groups. 111In-platelet counts in the infarct were reduced significantly by 64% in the ENOX group as compared to CON, and to a lesser extent (42%, n.s.) in the ENOX + SK group, but were not reduced by HEP and SK treatments. Neutrophil accumulation in the infarcts was decreased significantly and by more than 75% in the ENOX and ENOX + SK groups versus CON, but not in the HEP and SK groups. Also, only ENOX (10-100 micrograms/ml) significantly inhibited platelet-neutrophil rosettes formation in a plasmatic milieu. CONCLUSIONS: The ENOX treatment, as opposed to that of HEP, reduces myocardial platelet and neutrophil accumulations, and limits infarct size when given just before and during reperfusion. The benefits of ENOX on infarct size were not modified by SK, and may be related, at least in part, to an interaction with P-selectin-mediated cell adhesion.

Phase II trial of docetaxel with dexamethasone premedication in patients with advanced non-small cell lung cancer: the Canadian experience.

We performed a Phase II trial to evaluate the activity and tolerability of docetaxel as a single agent in the treatment of advanced non-small cell lung cancer (NSCLC). Forty-four patients with metastatic and/or locally advanced NSCLC received i.v. docetaxel 100 mg/m2 every 3 weeks for a median of 4 (range 1-11) cycles. All patients received premedication with oral dexamethasone 8 mg twice daily for 5 days starting the day before chemotherapy. Seven partial responses were observed among 35 evaluable patients, and the overall response rate was 20% (95% CI 8-37). The median response duration was 5 months, median survival time was 10 months and the estimated 1-year survival rate was 42%. Treatment was generally well tolerated. Febrile neutropenia occurred in 10 patients (23%); neutropenic infection occurred in 4 patients, and led to 2 toxic deaths (both patients had borderline exclusion criteria). The corticosteroid premedication effectively reduced the overall incidence (34%) and severity (4% severe) of fluid retention, and delayed the median time to onset from cycle 4 to cycle 7. This study shows the promising efficacy of docetaxel as monotherapy in advanced NSCLC, and combination chemotherapy regimens incorporating docetaxel are now being evaluated in this clinical setting.

Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5.

Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene. ATR1 encodes a protein of 692 amino acids, while ATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon. Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons. The two Arabidopsis reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution. A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1. The cDNA open reading frames were expressed in yeast. The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome c reductase activity. To gain more insight into the respective functions of the two reductases, the Arabidopsis cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast. Biochemical characterization of the Arabidopsis ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related Arabidopsis reductases similarly support the first oxidative step of the phenylpropanoid general pathway.

Docetaxel/doxorubicin/cyclophosphamide in the treatment of metastatic breast cancer.

Preliminary results from phase I trials suggest that the use of docetaxel (Taxotere) and doxorubicin (Adriamycin) is a well tolerated and highly active combination regimen for patients with metastatic breast cancer. The maximum tolerated dose of this combination was 50 mg/m2 of doxorubicin given as an intravenous bolus followed 1 hour later with 75 mg/m2 of docetaxel given as a 1-hour intravenous infusion. Because cardiotoxicity was not observed with this combination, we added cyclophosphamide (Cytoxan, Neosar) in a phase II trial to determine the antitumor activity and tolerability of this 3-drug combination as first-line therapy in patients with metastatic breast cancer. Preliminary results from this study indicate that the Taxotere/ Adriamycin/Cyclophosphamide (TAC) combination produces response rates of up to 80%. However, frequent grade 4 neutropenia was seen in 68% of cycles, febrile neutropenia in 5.5% of cycles, and grade 3 to 4 infection in .8% of cycles. Cardiac toxicity was rare, with 1 case of reversible congestive heart failure (2%), which occurred 2 months after completion of chemotherapy. These preliminary data show that TAC is highly active and that docetaxel did not significantly increase the cardiotoxicity of doxorubicin. Phase III studies in both the first-line and adjuvant settings are warranted.

Docetaxel/doxorubicin/cyclophosphamide in the treatment of metastatic breast cancer.

A pilot phase II study examined the feasibility of 75 mg/m2 of docetaxel (Taxotere) in combination with 50 mg/m2 of doxorubicin and 500 mg/m2 of cyclophosphamide (Cytoxan, Neosar) in the first-line treatment of metastatic breast cancer. This study was designed to evaluate the efficacy and toxicity of the docetaxel/doxorubicin/cyclophosphamide combination both alone and as induction before high-dose chemotherapy, supplemented by autologous peripheral blood stem-cell transplantation. Patients were divided into three groups: one group received 8 courses of docetaxel/doxorubicin/cyclophosphamide; the second received 4 to 6 courses of the same combination with cell sampling, followed by high-dose chemotherapy with autologous peripheral blood stem-cell transplantation; and the third group's regimen was identical to that of the second, with additional granulocyte-colony stimulating factor (G-CSF, filgrastim [Neupogen]). Of 28 patients (149 courses) evaluable for toxicity and response, the overall response rate was 82%, with 5 (18%) complete responses and 18 (64%) partial responses. The most frequent hematologic toxicity was neutropenia; grade 4 neutropenia occurred in 86% of patients, with febrile neutropenia in 9 patients (18%). There was no incidence of infection, possibly because of the administration of oral ciprofloxacin (Cipro) from days 5 to 15 of each cycle. Nonhematologic adverse events were not severe; there was no significant cardiotoxicity. Future randomized trials of docetaxel/doxorubicin/cyclophosphamide as first-line adjuvant therapy of high-risk patients and as induction chemotherapy are in development.

Prevention of venous thromboembolism after knee arthroplasty. A randomized, double-blind trial comparing enoxaparin with warfarin.

OBJECTIVE: To compare the effectiveness and safety of fixed-dose enoxaparin and adjusted dose warfarin in preventing venous thromboembolism after knee arthroplasty. DESIGN: A randomized, double-blind controlled trial. SETTING: 8 university hospitals. PATIENTS: 670 consecutive patients who had knee arthroplasty. INTERVENTION: Patients were randomly assigned to receive enoxaparin (30 mg subcutaneously every 12 hours) or adjusted-dose warfarin (international normalized ratio, 2.0 to 3.0). Both regimens were started after surgery. MEASUREMENTS: The primary end point was the incidence of deep venous thrombosis in patients with adequate bilateral venograms; the secondary end point was hemorrhage. RESULTS: Among the 417 patients with adequate venograms, 109 of 211 warfarin recipients (51.7%) had deep venous thrombosis compared with 76 of 206 enoxaparin recipients (36.9%) (P = 0.003). The absolute risk difference was 14.8% in favor of enoxaparin (95% Cl, 5.3% to 24.1%) Twenty-two warfarin recipients (10.4%) and 24 enoxaparin recipients (11.7%) had proximal venous thrombosis (P>0.2). The absolute risk difference was 1.2% in favor of warfarin (Cl, -7.2% to 4.8%). The incidence of major bleeding was 1.8% (6 of 334 patients) in the warfarin group and 2.1% (7 of 336 patients) in the enoxaparin group (P>0.2). The absolute risk difference was 0.3% in favor of warfarin (Cl, -2.4% to 1.8%). CONCLUSIONS: A postoperative, fixed-dose enoxaparin regimen is more effective than adjusted-dose warfarin in preventing deep venous thrombosis after knee arthroplasty. No differences were seen in the incidence of proximal venous thrombosis or clinically overt hemorrhage.

Angiosarcomas of the interatrial septum mimicking atrial myxomas.

Angiosarcoma of the heart is a rare tumor. This tumor is most frequently located in the right atrium and pericardium. Localization of a tumor in the interatrial septum usually suggests atrial myxoma. We report two cases of angiosarcoma originating from the interatrial septum, one extending into the right atrium and the other into the left atrium, mimicking atrial myxomas. Transesophageal echocardiography allowed the diagnosis and comprehensive assessment of compromised structures.

Docetaxel in patients with metastatic breast cancer: a phase II study of the National Cancer Institute of Canada-Clinical Trials Group.

PURPOSE: The National Cancer Institute of Canada-Clinical Trials Group (NCIC-CTG) conducted a phase II study to assess the efficacy and toxicity of docetaxel as first-line chemotherapy in metastatic breast cancer (MBC). PATIENTS AND METHODS: Fifty-one patients with measurable MBC were studied. Three patients were ineligible and were excluded from analysis. The planned dose of docetaxel was 100 mg/m2 intravenously (i.v.) every 3 weeks. Prior adjuvant chemotherapy was allowed if at least 12 months had elapsed from completion of treatment to recurrence. RESULTS: The most severe toxicity was granulocytopenia. Ten patients (20.8%) were hospitalized for febrile neutropenia. The protocol was amended to a starting dose of 75 mg/m2 for the last 16 patients. Sixty percent of patients experienced hypersensitivity reactions (HSRs). After two protocol amendments, the use of a premedication regimen of oral dexamethasone and i.v. H1 and H2 blockers prevented significant HSRs. Edema developed in 62% of patients and was cumulative, was present in 50% who received greater than 400 mg/m2, and was not improved by premedication regimens. Following an independent radiology review, 22 partial remissions and four complete responses in 47 assessable patients were confirmed (response rate, 55%; 95% confidence interval [CI], 40% to 69%). The response rate for 15 assessable patients registered at 75 mg/m2 was 40% (95% CI, 16% to 67%); for 32 assessable patients registered at 100 mg/m2, the response rate was 63%, (95% CI, 43% to 78%). CONCLUSION: Docetaxel is an active agent in MBC. Its activity as a single agent is comparable to many combination chemotherapy regimens and is not affected by prior adjuvant chemotherapy. Studies are ongoing to improve its therapeutic index and to incorporate docetaxel in combination chemotherapy regimens.

The effects of spiramycin and/or scaling on advanced periodontitis in humans.

It has long been questioned whether antibiotics, used as a supplement to traditional therapy, provide any lasting benefit in the treatment of chronic periodontitis. This study was designed to evaluate Spiramycin as an adjunct to scaling and root planing in the treatment of advanced chronic periodontitis. In total, 193 patients with advanced periodontitis were recruited in seven centres using selection criteria previously described. After undergoing thorough scaling and root planing, all patients randomly received either Spiramycin, 1,500,000 international units, twice per day (IU, bid) for 14 days (96 patients), or a visually-identical placebo capsule (97 patients). The clinical parameters measured were plaque index, crevicular fluid level, probing depths, bleeding on probing and attachment level changes. Data was recorded at baseline, two-, eight-, 12- and 24-weeks visits. A total of 189 patients completed the study (96 placebo, 93 Spiramycin). Statistically significant differences in probing depth, favoring Spiramycin, were seen at two weeks (p < 0.0125), eight weeks (p < 0.0020), 12 weeks (p < 0.0032) and 24 weeks (p < 0.0075). Spiramycin also produced a significant improvement in attachment level at 12 weeks (p < 0.0146). All other clinical parameters showed no difference between drug and placebo. This study shows that Spiramycin, as an adjunct to thorough scaling and root planing, provides a statistically significant improvement in probing depths for up to 24 weeks when compared with scaling and root planing alone. Both longer studies and microbiologic evaluations are necessary to determine whether a more lasting benefit is possible.

The low molecular weight heparin Enoxaparin inhibits the consumption of factor VII and prothrombin activation in vivo associated with elective knee replacement surgery.

Patients over 40 years of age who undergo elective orthopaedic surgery have a relatively high risk for developing post-surgical deep vein thrombosis (DVT). Prophylactic use of heparin or low molecular weight heparins can reduce the incidence of post-operative DVT by up to 80%. It is not known whether prophylaxis is achieved by inhibition of prothrombin activation or catalysis of thrombin inhibition in vivo. We determined the changes in concentrations of factor VII zymogen and thrombin-antithrombin III (the latter as an index of prothrombin activation) in the plasmas of 129 patients randomized to receive two daily subcutaneous injections of placebo or 30 mg of Enoxaparin after elective knee surgery. Enoxaparin reduced the frequency of post-surgical DVT by 70%. The concentration of factor VII zymogen had decreased by approximately 50% within 24 h after the knee surgery, followed by a gradual increase to near presurgical values. Additionally, post-Enoxaparin plasmas had statistically significant higher concentrations of factor VII zymogen than post-placebo plasmas. Post-Enoxaparin plasmas had significantly lower concentrations of endogenous thrombin-antithrombin III than comparable post-placebo plasmas. Finally, post-Enoxaparin plasmas inactivated exogenous factor Xa and thrombin more effectively than comparable post-placebo plasmas. As Enoxaparin moderated the generation of endogenous thrombin-antithrombin III after elective knee surgery, inhibition of prothrombin activation in vivo by Enoxaparin may be important for its prophylactic antithrombotic effect.

Prevention of deep vein thrombosis after major knee surgery--a randomized, double-blind trial comparing a low molecular weight heparin fragment (enoxaparin) to placebo.

Consecutive patients undergoing knee arthroplasty or tibial osteotomy at four participating hospitals received either enoxaparin, 30 mg subcutaneously every 12 h (n = 66) or an identical-appearing placebo (n = 65). All study medications started the morning after the operation and were continued up to a maximum of 14 days. Patients underwent surveillance with 125I-fibrinogen leg scanning and impedance plethysmography. Bilateral contrast venography was performed routinely at Day 14 or at time of discharge, if sooner. Deep vein thrombosis was detected by venography in 35 of 54 patients (65%) in the placebo group and in 8 of 41 patients in the enoxaparin group (19%), a risk reduction of 71%, P less than 0.0001. For the entire study group, deep vein thrombosis was detected by either venography of non-invasive tests in 37 of 64 patients (58%) in the placebo group and in 11 of 65 patients (17%) in the enoxaparin group, a risk reduction of 71%, P less than 0.0001. Proximal vein thrombosis was found in 19% of the placebo patients and in none of the enoxaparin patients, a risk reduction of 100%, P less than 0.001. Bleeding complications occurred in 5 of 65 patients (8%) in the placebo group and in 4 of 66 patients (6%) in the enoxaparin group, P = 0.71. There were no differences in the amount of blood loss, minimum hemoglobin levels and number of units of packed red cells given between the two treatment groups. We conclude that a fixed dose regimen of enoxaparin, started post-operatively, is an effective and safe regimen for reducing the frequency of deep vein thrombosis after major knee surgery.

Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense.

The complete nucleotide sequence of the open reading frame (ORF) located upstream of the glnA structural gene for glutamine synthetase (GS) in Azospirillum brasilense Sp7 was determined. This ORF, which codes for a 12 kDa protein, was identified as glnB, the structural gene for the PII protein, a component of the adenylylation cascade involved in the regulation of GS activity in some gram-negative bacteria. Transcription analysis and mRNA mapping of glnB and glnA of A. brasilense was performed with bacteria grown under different physiological conditions. The glnA gene can be transcribed either as a glnB-A mRNA of 2.4 kb or as a glnA mRNA of 1.5 kb. Differential expression of the two mRNAs was found to depend on the nitrogen source. The glnB-A mRNA was the major transcript under nitrogen fixation conditions, while the synthesis of the glnA mRNA was almost completely abolished. The glnA mRNA was predominantly produced in NH4(+)-containing medium. Transcription start site analysis revealed the presence of three different types of nitrogen-regulated promoters. GlnB-A mRNA was transcribed selectively from tandem promoters. One of them is similar to the NtrA-dependent promoter and the other to the Escherichia coli sigma 70 promoter. The synthesis of glnA mRNA was regulated by a promoter, which was repressed (or non-activated) only under conditions of nitrogen fixation, when moleuclar nitrogen was the sole nitrogen source. The transcriptional initiation site in front of glnA is not preceded by a canonical E. coli sigma 70 promoter. A sequence reminiscent of the NtrA-dependent promoter consensus, except for a fundamental mismatch, was found at positions -33 to -21. This sequence overlapped a putative "weak" NtrC-binding site, similar to those identified in enteric bacteria. From these results, it is postulated that glnA mRNA is controlled by a novel type of nitrogen-regulated promoter.

Regulation of transcription and promoter mapping of the structural genes for nitrogenase (nifHDK) of Azospirillum brasilense Sp7.

Transcription of the structural genes for nitrogenase (nifHDK) in Azospirillum brasilense Sp7 was analysed using Northern blots of total RNA extracted from cultures grown under nitrogen-fixing conditions. Hybridization with an internal nifH probe revealed two transcripts, a major one (by concentration) of 1.1 kb corresponding to nifH and a minor one of 5.6 kb corresponding to nifHDK. Hybridization with nifD or nifK probes revealed the minor transcript of 5.6 kb. This confirms that the nifHDK genes are organized as a single transcription unit and suggests regulation at the level of termination of transcription. The complete nucleotide sequence of nifH was established and the DNA region upstream of the initiation codon was analysed for transcription and translation signals. The nifH open reading frame (ORF) is preceded by an NtrA-dependent promoter and two elements homologous to upstream activator sequences (UAS) required for NifA-mediated activation in other diazotrophs. Promoter mapping with S1 nuclease revealed two start sites located 10 bp and 40 bp downstream of the NtrA-dependent promoter.

In vitro susceptibilities of oral bacterial isolates to spiramycin.

Four hundred strains of oral bacteria were tested for their susceptibility to spiramycin. Actinobacillus actinomycetemcomitans and most species of Lactobacillus were resistant to the antibiotic. All strains of cariogenic Streptococcus mutans and most strains of bacterial species implicated in adult chronic periodontitis (Bacteroides gingivalis, B. intermedius, and Treponema denticola) were susceptible to spiramycin.

Effects of adjunctive treatment of periodontitis with tetracycline and spiramycin.

The present study was undertaken to compare the efficacy of two antibiotics, spiramycin and tetracycline, with a placebo when used adjunctively with scaling and root planing in the treatment of advanced adult chronic periodontitis. This was a double-blind, parallel, randomized trial with one factor (drug) at three levels. Ninety-six patients (mean age 46 +/- 1) were randomly assigned into one of three groups. All groups were scaled and root planed with each respective group receiving either spiramycin, tetracycline, or a placebo for 2 weeks. Two sites with probing depth of at least 7 mm were evaluated and the following clinical parameters were measured at baseline, 2, 8, 12, and 24 weeks: plaque index, bleeding on probing, crevicular fluid, probing depth, and change in the attachment level. The changes in the subgingival bacteria were monitored also using a differential staining technique. Seventy-nine patients completed the study. At the end of 24 weeks, although all three groups had shown clinical improvement when compared to the baseline data, there were no significant intergroup differences in any of the clinical parameters measured. While the proportion of spirochetes were significantly decreased (P less than 0.05) at 2- and 8-week intervals in both tetracycline and spiramycin groups (26% to 0.04% and 28% to 0.04%, respectively), compared to the placebo group (30% to 7%), only in the spiramycin group was the proportion of spirochetes significantly lower than the placebo group at the 24-week interval (3% and 11%, respectively). At week 24, the proportion of spirochetes in the tetracycline group had rebounded to 7%, which was not significantly different from the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7.

A 30 kb DNA region from Azospirillum brasilense Sp7, containing the nitrogenase structural genes (nifHDK), has been cloned. The presence of nif genes, in the 20 kb located next to nifHDK, was explored by Tn5 mutagenesis after subcloning various restriction fragments in the broad-host-range suicide vehicle pSUP202. Over 25 mutations due to Tn5 random insertions were obtained in the 20 kb and each recombined into the genome of strain Sp7. Four new nif loci were identified, located at about 4, 9, 12 and 18 kb downstream from nifK respectively. Hybridization with heterologous nif probes from Klebsiella pneumoniae, Bradyrhizobium japonicum and Azorhizobium caulinodans was performed to characterize the new nif regions. The region proximal to nifK appears to contain nifE and the region distal to nifK contains genes homologous to nifUS and fixABC. nifgene(s) from the fourth locus were not identified. Mutants in this locus, which were devoid of nitrogenase activity when tested under nitrogen-free conditions, displayed a high nitrogenase activity when glutamate was added to the growth medium. This phenomenon was also observed with mutants of the fixABC homology region, but to a lesser extent. Homology between strain Sp7 total DNA and a nifB-containing probe from B. japonicum was detected, although the hybridizing region was not part of the nif cluster described above.