Betaproteobacteria are a key component of surface water biofilters that maintain sustained manganese removal in response to fluctuations in influent water temperature.

The health risks associated with manganese (Mn) in drinking water, and an improved understanding of Mn accumulation within, and subsequent release from, distribution systems, have increased the need for robust, sustainable treatment options to minimize Mn concentrations in finished water. Biofiltration is an established and effective method to remove Mn in groundwater however, Mn removal in surface water biofilters is an emerging treatment process that has not been extensively studied. Seasonal variations in water temperature can present an operational challenge for surface water biofilters which may see reduced Mn removal under colder conditions. This study examined the microbiomes of surface water biofilters at three utilities (ACWD WTP, WTP B, and WTP D) which all experienced similar seasonal fluctuations in influent water temperature. High Mn removal was observed at the ACWD WTP for much of the year, but Mn removal decreased with a concurrent decrease in the influent water temperature (58% ± 22%). In contrast, both WTP B and WTP D achieved year-round Mn removal (84% ± 5% and 93% ± 8% respectively). Marker gene (16S rRNA) sequencing analysis of the biofilter microbiomes identified a high abundance of Betaproteobacteria in WTP B and WTP D (37% ± 12% and 21% ± 3% respectively), but a low abundance of Betaproteobacteria in the ACWD WTP (2% ± 2%). The microbiomes of new bench-scale biofilters, in operation at the ACWD WTP, were also investigated. The abundance of Betaproteobacteria was significantly greater (p < 0.05) after the biofilters had acclimated than before acclimation, and differential abundance analysis identified 6 genera within the Betaproteobacteria class were enriched in the acclimated microbiome. Additionally, the acclimated biofilters were able to maintain high Mn removal performance (87% ± 10%) when the influent water temperature decreased to 10 °C or less. Further analysis of previously published studies found the abundance of Betaproteobacteria was also significantly greater (p < 0.001) in biofilters with sustained Mn removal than in biofilters which did not treat for Mn as a contaminant, despite differences in design scale, source water, and media type. Microbiome network analysis identified multiple co-occurrence relationships between Betaproteobacteria and Mn oxidizing bacteria in the WTP B and WTP D biofilters, suggesting indirect contributions by Betaproteobacteria to biological Mn oxidation. These co-occurrence relationships were not present in the full-scale ACWD WTP microbiome. Whether the role of Betaproteobacteria in biological Mn oxidation is direct, indirect, or a combination of both, they are consistently present at a high abundance in both groundwater and surface water biofilters with sustained Mn removal, and their absence may contribute to the seasonal fluctuations in Mn removal observed at the ACWD WTP. This new insight to Betaproteobacteria and their role in Mn biofiltration could contribute to water innovation and design that would improve the reliability of Mn removal.

Chemical recovery and browning of Nova Scotia surface waters in response to declining acid deposition.

Declining emissions of sulfur and nitrogen have curtailed acid deposition across large areas of North America and Europe. This has allowed many lakes to recover from acidification, with decreases in sulfate, increases in pH, and increases in alkalinity. But reduced acid deposition has not always coincided with chemical lake recovery. Surface waters in Nova Scotia did not exhibit clear evidence of recovery as recently as 2007, due in part to increasing organic acidity and slow replenishment of base cations. In an updated assessment with data collected as recently as 2019, we analyze water chemistry representing 81 lakes and rivers and two precipitation monitoring stations over up to 41 years. We find that Nova Scotia surface waters are now exhibiting signs of chemical recovery. We estimated the linear decrease in precipitation sulfate and nitrate yield at up to 0.31 and 0.18 kg ha-1 year-2, respectively, and the linear increase in precipitation pH at up to 0.014 year-1. Sulfate decreased in 60 of 62 lakes and 14 of 17 rivers (-0.0051 to -0.23 mg L-1 year-1), while pH increased in 55 of 64 lakes and 11 of 17 rivers (0.0015-0.072 year-1). Apparent colour increased in 54 of 62 lakes and 13 of 17 rivers (0.0026-3.9 Pt-Co year-1). We identified increasing aluminum trends in 46 of 61 lakes, and we show using size-exclusion chromatography that binding to organic and iron-based colloids may help to explain these trends. To the extent that increases in apparent colour are explained by chromophoric dissolved organic matter (DOM), they imply greater binding capacity for metals in surface waters, and greater capacity for DOM to stabilize metal (oxyhydr)oxide colloids.

Linear versus volumetric CT analysis in predicting tension-free fascial closure in abdominal wall reconstruction.

BACKGROUND: Improved outcomes of abdominal wall reconstruction (AWR) have been shown when tension-free fascial closure (TFFC) is achieved. Our objective was to determine the clinical and radiologic predictors of TFFC in patients undergoing AWR. STUDY DESIGN: We conducted a single institution retrospective cohort study of adults who underwent AWR between 2007 and 2018. Demographics, hernia characteristics and operative data were collected. Linear and volumetric variables were obtained from preoperative abdominal CT scans, the latter following 3D reconstruction. Logistic regression was used to evaluate predictors of TFFC. Area under the curve (AUC) ≥ 0.70 was considered to have acceptable discrimination. RESULTS: A total of 108 patients were eligible for analysis. The mean age was 57 ± 11 years and 53 (49%) were female. 42 (39%) hernias were recurrent, 10 (9%) patients had a stoma and 9 (8%) had a history of open abdomen. The mean defect width was 11 ± 4 cm and mean defect surface area was 150 ± 95 cm2. The most common AWR technique was endoscopic component separation 75 (69%). TFFC was achieved in 90 (83%) patients. No demographics or 3D volumetric measures were predictive of TFFC (all AUC < 0.7). European hernia society (EHS) class M1 was predictive of failure of TFFC [AUC = 0.70; odds ratio 7.0 (referent M3); 95% confidence interval, 2.1-23.8]. Linear variables of rectus muscle separation were the most predictive of TFFC (AUC 0.73-0.77). CONCLUSION: In contrast to clinical characteristics, radiologic characteristics of large incisional hernias requiring AWR are predictive of TFFC. In particular, EHS class M1 and linear variables of rectus muscle separation appear to be better predictors of TFFC than volumetric measurements.

Biological and physico-chemical mechanisms accelerating the acclimation of Mn-removing biofilters.

This study investigated treatment strategies which accelerated the acclimation of new Mn-removing biofilters to help utilities respond to changing Mn regulations, such as the recent introduction of a health-based maximum acceptable concentration and a reduction in the aesthetic objective for Mn in drinking water by Health Canada. Bench-scale filters of either GAC or anthracite media were fed with applied water containing Mn (17-61 µg/L) from a full-scale plant over 294 days. Treatment strategies included the addition of H2O2 (1 mg/L) and/or an increase in pH from 6.8 to 7.5 through the addition of NaOH. The potential physico-chemical and biological mechanisms responsible for accelerated biofilter acclimation under the various redox conditions were investigated through thermodynamic modelling, to predict homogeneous Mn oxide formation, and 16S rRNA gene amplicon sequencing, to characterize the microbial community within the filters. GAC filters treated with NaOH, and both H2O2 and NaOH, were the first to acclimate (< 20 µg/L Mn in filter effluent) after 59 and 63 days respectively, while the ambient GAC filter took almost 3 times as long to acclimate (168 days), and the anthracite filters which received the same chemically adjusted water took almost 4 times as long (226 and 251 days, respectively). The accelerated acclimation in the treated GAC filters was likely due to physico-chemical oxidation via three potential mechanisms: (1) homogeneous oxidation of dissolved Mn(II) to Mn(III)/Mn(IV) oxides and the subsequent removal of oxides from solution through adherence to the GAC surface, (2) adsorption of dissolved Mn(II) to GAC and subsequent homogeneous or biological oxidation, or (3) formation of colloidal Mn(III)/Mn(IV) oxides and subsequent adsorption of dissolved Mn(II) to the Mn colloids. In the untreated GAC filter and all anthracite filters, which did not benefit from improved redox conditions or an active surface, physico-chemical mechanisms alone were insufficient for consistent Mn removal to less than 20 µg/L. Acclimation in these filters was delayed until a microbiome enriched with bacteria capable of biological nitrification and Mn oxidation evolved within the filters. The acclimated microbiome was consistent between GAC and anthracite filters and was significantly different from the non-acclimated microbiome (p < 0.001) initially formed during the early operation of the filters. Interestingly, treatment with NaOH, and NaOH and H2O2, which accelerated physico-chemical oxidation in GAC filters, was observed to delay the development of biological oxidation in anthracite filters, and thus deferred acclimation. Although some filters took longer to acclimate than others, once acclimation was reached all filters had a similar microbiome and were able to consistently remove Mn to below 20 µg/L.

Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro.

BACKGROUND AND OBJECTIVE: The therapeutic benefits of platelet-rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. MATERIAL AND METHODS: The levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium- and thrombin-activated PRP samples from five donors were quantified by enzyme-linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody-coated beads on HUVEC proliferation was also tested. RESULTS: Average concentrations of VEGF, PDGF-BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody-coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose-dependent manner. CONCLUSION: This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.

Detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment.

BACKGROUND/AIM: Although the role of bacteria in the etiology of periodontitis is well established, it has been suggested that herpetic viruses could contribute to the initiation and progression of this disease. The aim of this study was to determine the prevalence of human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) in gingival crevicular fluid (GCF) samples obtained from periodontally healthy, gingivitis and periodontitis patients. In addition, the effect of periodontal treatment (scaling and root planing) on the persistence of herpetic viruses was evaluated in a sub-group of patients suffering from chronic periodontitis. METHODS: The presence of viruses in GCF samples was assessed by a nested PCR amplification technique. The persistence of viruses in periodontal sites was evaluated following a scaling and root planing therapy. RESULTS: A statistically significant higher prevalence of HCMV was observed in periodontitis patients as compared to healthy control subjects (35 vs. 8%, respectively; P = 0.0377). A trend for a higher prevalence of HSV was also noted in the periodontitis group, in comparison with healthy control subjects. In addition, a higher prevalence of HCMV was associated with deep periodontal pockets in subjects suffering from periodontitis. In the sub-group of periodontitis patients, periodontal therapy resulted in the elimination (HCMV and EBV) or reduction (HSV) of the herpetic viruses. CONCLUSIONS: This study showed that the prevalence of HCMV and HSV viruses in GCF is higher in patients suffering from periodontitis compared to periodontally healthy subjects, and that the prevalence of HCMV is higher in deep periodontal pockets. It also brought evidences that periodontal therapy may be associated with virus elimination in diseased sites.

Effect of coagulation and flocculation conditions on water quality in an immersed ultrafiltration process.

The removal of natural organic matter under variable coagulation and flocculation pretreatment conditions was evaluated for three surface waters in an immersed ultrafiltration (UF) process. Coagulation with alum, flocculation and UF treatment were conducted in a bench-scale test apparatus designed to simulate pilot- and full-scale water treatment systems. Variable coagulation and flocculation operating conditions were investigated, including coagulant dose, hydraulic retention time (HRT) and mixing intensity (e.g. velocity gradient). Treatment performance was evaluated by measuring specific water quality parameters in the permeate stream, including dissolved organic carbon (DOC), UV254 and true colour. Coagulant dose was found to be the most important variable for treatment performance with regard to permeate water quality, with significantly lower alum dosages required to achieve enhanced coagulation water quality targets than conventional filtration systems. Experiments conducted to evaluate variable flocculation stage HRT and applied velocity gradient demonstrated that traditional set points for these operating variables, applied in conventional filtration systems, may not be required in UF systems. In particular, optimized UF permeate water quality was found with reduced flocculation retention times (e.g. <10 minutes) and mixing intensities (e.g. < 100 s(-1)). The impact of intermittent air scour, or air sparging, operations in the UF process tank during operation was also evaluated. The use of air scour, tested as an intermittent operation at an applied velocity gradient of 50 s(-1) was found to significantly reduce DOC concentrations and UV254 measurements in the UF permeate stream when compared with UF operations without air scour.

Sequential UV- and chlorine-based disinfection to mitigate Escherichia coli in drinking water biofilms.

This study was designed to examine the potential downstream benefits of sequential disinfection to control the persistence of Escherichia coli under conditions relevant to drinking water distribution systems. Eight annular reactors (four polycarbonate and four cast iron) were setup in parallel to address various factors that could influence biofilm growth in distribution systems. Eight reactors were treated with chlorine, chlorine dioxide and monochloramine alone or in combination with UV to examine the effects on Escherichia coli growth and persistence in both the effluent and biofilm. In general, UV-treated systems in combination with chlorine or chlorine dioxide and monochloramine achieved greater log reductions in both effluent and biofilm than systems treated with chlorine-based disinfectants alone. However, during UV-low chlorine disinfection, E. coli was found to persist at low levels, suggesting that the UV treatment had instigated an adaptive mutation. During UV-chlorine-dioxide treatment, the E. coli that was initially below the detection limit reappeared during a low level of disinfection (0.2 mg/L) in the cast iron systems. Chloramine was shown to be effective in disinfecting suspended E. coli in the effluent but was unable to reduce biofilm counts to below the detection limit. Issues such as repair mechanism of E. coli and nitrification could help explain some of these aberrations. Improved understanding of the ability of chlorine-based disinfectant in combination with UV to provide sufficient disinfection will ultimately effect in improved management and safety of drinking water.

Biostability and disinfectant by-product formation in drinking water blended with UF-treated filter backwash water.

The overall objective of this study was to investigate the impact of blending membrane-treated water treatment plant (WTP) residuals with plant-filtered water on finished water quality in terms of biostability and disinfectant by-product (DBP) formation. Filter backwash water (FBWW) was treated with a pilot-scale ultrafiltration (UF) membrane to produce permeate that was blended with plant-finished water. The batch studies involved storing samples for a specified time with a disinfectant residual to simulate residence time in the distribution system. Both chlorinated and non-chlorinated FBWW streams were evaluated, and the experimental design incorporated free chlorine, monochloramine, and chlorine dioxide in parallel to a model system that did not receive a disinfectant dose. The results of the study found that blending 10% UF-treated FBWW with plant-filtered water did not have an impact on water biostability as monitored with heterotrophic plate counts (HPCs) or DBP concentrations as monitored by TTHM and HAA5 concentrations. However, the presence of preformed THM and HAA species found in chlorinated FBWW streams may result in higher levels of initial DBP concentrations in blended water matrices, and could have a significant impact on finished water quality in terms of meeting specific DBP guidelines or regulations.

A field study evaluation for mitigating biofouling with chlorine dioxide or chlorine integrated with UV disinfection.

The drinking water industry is continually seeking innovative disinfection strategies to control biofouling in transmission systems. This research, conducted in collaboration with the East Bay Municipal Utility District (EBMUD) in California, compared the efficacy of chlorine dioxide (ClO2) to free chlorine (Cl2) with and without pre-treatment with low-pressure ultraviolet (UV) light for biofilm control. An additional goal was to determine disinfection by-product (DBP) formation with each disinfection strategy. Annular reactors (ARs) containing polycarbonate coupons were used to simulate EBMUD's 90-mile aqueduct that transports surface water from a source reservoir to treatment facilities. ARs were dosed with chemical disinfectants to achieve a residual of 0.2 mg/L, which is a typical value mid-way in the aqueduct. The experiment matrix included four strategies of disinfection including UV/ClO2, ClO2, UV/Cl2 and Cl2. Two ARs acted as controls and received raw water (RW) or UV-treated water. The data presented show that the UV/ClO2 combination was most effective against suspended and attached heterotrophic (heterotrophic plate count, HPC) bacteria with 3.93 log and 2.05 log reductions, respectively. ClO2 was more effective than Cl2 at removing suspended HPC bacteria and similarly effective in biofilm bacterial removal. UV light alone was not effective in controlling suspended or biofilm bacteria compared to treatment with ClO2 or Cl2. Pre-treatment with UV was more effective overall for removal of HPC bacteria than treating with corresponding chemical disinfectants only; however, it did not lower required chemical dosages. Therefore, no significant differences were observed in DBP concentrations between ARs pre-treated with UV light and ARs not pre-treated. Disinfection with ClO2 produced fewer total trihalomethanes (TTHMs) and haloacetic acids (HAAs) than chlorination but did produce low levels of chlorite. These data indicate that replacing Cl2 with ClO2 would further control microbiological re-growth and minimize TTHM and HAA formation, but may introduce other DBPs.

Association of granulocytosis with poor prognosis in patients with acute myelogenous leukemia and translocation of chromosomes 8 and 21.

Forty-three patients with acute myelogenous leukemia (AML) and a translocation 8;21 were reviewed. The patients' median age was 30 years, and 62% were men. Twenty-three patients (53%) had loss of a sex chromosome and ten (23%) had other chromosomal abnormalities in addition to the 8;21 translocation. Complete remission (CR) with induction chemotherapy was achieved in 40 patients (93%). The median CR duration was 18 months (range, 1 to 137+ months). Median survival time was 17 months (range, 0.5 to 138+ months) with a 3-year survival rate of 31%. Twenty-three patients (53%) relapsed between 1 and 58 months after entering remission with reinduction therapy resulting in 8 CRs (35%). Thirteen characteristics were examined for an effect on survival. The most striking finding was a significant association between elevated absolute granulocyte count and poor survival (P = .002). WBC count greater than 10,000/microL was also associated with shorter survival (P = .05). Patient age, albumin level, and platelet count showed trends for survival association. Although patients with AML and t(8;21) are regarded as a favorable group with respect to survival, we found a subset of patients who do very poorly. Intensive or more investigational approaches in first remission should be considered for these patients.

Platelet-rich plasmas: growth factor content and roles in wound healing.

UNLABELLED: Platelet-rich plasmas (PRPs) are used in a variety of clinical applications, based on the premise that higher growth factor content should promote better healing. In this study, we have determined the effects of calcium and thrombin on the release of EGF, TGF-alpha, IGF-1, Ang-2 and IL-1beta from PRPs, and assessed the mitogenic potential of PRP supernatants on osteoblast and endothelial cell division. ELISA assays indicate that (i) mean growth factor concentrations vary from traces (TGF-alpha) to 5.5 ng/mL (IGF-1), (ii) there are significant variations in growth factor concentrations between individuals, and (iii) calcium and thrombin regulate growth factor release, synthesis, and/or degradation in stereotyped patterns that are specific to each growth factor. PRP supernatants promote strong osteoblast and endothelial cell divisions, supporting the concept that PRPs may be beneficial in wound healing. ABBREVIATIONS: PRPs, platelet-rich plasmas; GFs, growth factors; EGF, epidermal growth factor; TGF-alpha, transforming growth factor-alpha; IGF-1, insulin-like growth factor-1; Ang-2, angiopoietin-2; IL-1beta, interleukin-1 beta; HUVECs, human umbilical vein endothelial cells; hFOB 1.19, human fetal osteoblasts; and FBS, fetal bovine serum.

Disinfectant efficacy of chlorite and chlorine dioxide in drinking water biofilms.

The drinking water industry is closely examining options to maintain disinfection in distribution systems. In particular this research compared the relative efficiency of the chlorite ion (ClO2-) to chlorine dioxide (ClO2) for biofilm control. Chlorite levels were selected for monitoring since they are typically observed in the distribution system as a by-product whenever chlorine dioxide is applied for primary or secondary disinfection. Previous research has reported the chlorite ion to be effective in mitigating nitrification in distribution systems. Annular reactors (ARs) containing polycarbonate and cast iron coupons were used to simulate water quality conditions in a distribution system. Following a 4 week acclimation period, individual ARs operated in parallel were dosed with high (0.25mg/l) and low (0.1mg/l) chlorite concentrations and with high (0.5 mg/l) and low (0.25mg/l) chlorine dioxide concentrations, as measured in the effluent of the AR. Another set of ARs that contained cast iron and polycarbonate coupons served as controls and did not receive any disinfection. The data presented herein show that the presence of chlorite at low concentration levels was not effective at reducing heterotrophic bacteria. Log reductions of attached heterotrophic bacteria for low and high chlorite ranged between 0.20 and 0.34. Chlorine dioxide had greater log reductions for attached heterotrophic bacteria ranging from 0.52 to 1.36 at the higher dose. The greatest log reduction in suspended heterotrophic bacteria was for high dose of ClO2 on either cast iron or polycarbonate coupons (1.77 and 1.55). These data indicate that it would be necessary to maintain a chlorine dioxide residual concentration in distribution systems for control of microbiological regrowth.

Effect of disinfectants on microbial ecology in model distribution systems.

This research was conducted to assess the impact of various disinfectants on bacterial water quality within model distribution systems (i.e. annular reactors). After colonization with non-disinfected water, annular reactors were treated with relatively low doses of chlorine (0.4 mg/l), chlorine dioxide (0.15 mg/l), or chloramines (0.9 mg/l). Under the tested conditions, bacterial inactivation varied as a function of disinfectant type (ranking by efficiency per mg of oxidant: ClO2 > Cl2 > ClNH2) and sample type (bulk water vs. biofilm). Depending on the disinfectant, the log inactivation of suspended and attached bacteria were 0.7-1.2 and 0.5-1.0, respectively. The characterization of microbial communities in drinking water can be performed using biochemical and/or molecular methods. In this study, biochemical tests were used, showing that pseudomonad and pseudomonad-like bacteria, as in other studies, were the most predominant micro-organisms (e.g. Pseudomonas fluorescens, Brevundimonas vescularis). The ratio Gram-positive to Gram-negative organisms was 1 to 3. No drastic differences were observed between the non-treated and disinfected pipes. Based on the bacteriological data presented in these experiments, chlorine dioxide represents an alternative to chlorine for certain distribution systems.

An active atmospheric methane sink in high Arctic mineral cryosols.

Methane (CH4) emission by carbon-rich cryosols at the high latitudes in Northern Hemisphere has been studied extensively. In contrast, data on the CH4 emission potential of carbon-poor cryosols is limited, despite their spatial predominance. This work employs CH4 flux measurements in the field and under laboratory conditions to show that the mineral cryosols at Axel Heiberg Island in the Canadian high Arctic consistently consume atmospheric CH4. Omics analyses present the first molecular evidence of active atmospheric CH4-oxidizing bacteria (atmMOB) in permafrost-affected cryosols, with the prevalent atmMOB genotype in our acidic mineral cryosols being closely related to Upland Soil Cluster α. The atmospheric (atm) CH4 uptake at the study site increases with ground temperature between 0 °C and 18 °C. Consequently, the atm CH4 sink strength is predicted to increase by a factor of 5-30 as the Arctic warms by 5-15 °C over a century. We demonstrate that acidic mineral cryosols are a previously unrecognized potential of CH4 sink that requires further investigation to determine its potential impact on larger scales. This study also calls attention to the poleward distribution of atmMOB, as well as to the potential influence of microbial atm CH4 oxidation, in the context of regional CH4 flux models and global warming.

Phosphotransferase system of Streptococcus salivarius: characterization of the ptsH gene and its product.

The Streptococcus salivarius ptsH gene encoding histidine-containing phosphocarrier protein (HPr) of the phosphotransferase system (PTS) has been cloned, sequenced, and found to be part of a ptsH, ptsI operon. Upstream from ptsH, putative -35 and -10 boxes and a Shine-Dalgarno sequence highly similar to the Escherichia coli consensus regulatory elements were identified. A second promoter, located in the ptsH coding sequence was also observed and is sufficient for the expression of the S. salivarius ptsI gene, encoding enzyme I of the PTS in E. coli [Gagnon et al., Gene 121 (1992) 71-78]. The amino acid sequence of S. salivarius HPr, inferred from the ptsH sequence, shared identity varying between 37 and 76% with known HPr from other bacteria. Moreover, the S. salivarius HPr shared 78% identity with an HPr-like protein of Aspergillus fumigatus, a eukaroytic mold that does not possess a functional PTS. Expression analysis of S. salivarius HPr in E. coli demonstrated that (i) S. salivarius ptsH is expressed in E. coli under the control of its own promoter, (ii) S. salivarius HPr synthesized by E. coli is completely processed by methionine aminopeptidase, and (iii) S. salivarius HPr is phosphorylated in vivo by E. coli enzyme I. It was also observed that, in E. coli, the copy number of pUC18 bearing S. salivarius ptsH was reduced more than 25-fold, as compared to pUC18 without an insertion.

Cloning, sequencing and expression in Escherichia coli of the ptsI gene encoding enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system from Streptococcus salivarius.

We present the cloning and sequencing of the ptsI gene, encoding enzyme I (EI) of the phosphoenolpyruvate (PEP): sugar phosphotransferase (PTS) transport system from Streptococcus salivarius. The ptsI gene corresponds to an open reading frame of 1731 nucleotides, which translates into a putative 577-amino acid (aa) protein with a M(r) of 62,948 and a pI of 4.49. The EI was produced in Escherichia coli under the control of its own promoter located immediately upstream of ptsI, a situation never previously reported for any other gene coding for an EI. The deduced aa sequence of the S. salivarius EI shows a high degree of similarity with the E. coli EI and the EI moiety of the multiphosphoryl transfer protein from Rhodobacter capsulatus. The S. salivarius EI also shares a highly conserved aa cluster with a non-PTS protein, the maize pyruvate:orthophosphate dikinase. The conserved cluster is located in a domain which is hypothesized to be the PEP-binding site.

Cloning of hybridomas by a single-cell transfer technique.

A method for cloning hybridomas is described which involves transfer of single cells with a capillary tubing connected to a suction apparatus. This method enables the efficient recovery of antibody-producing clones of differing abundances from the parent hybridoma cultures. The 4 cell lines submitted to this cloning procedure produced a 100% yield of positive wells upon recloning by the same method.

Studies on the mechanism of action of various vasodilators.

1 The vascular relaxant effects of histamine, adenosine, isoprenaline nitroglycerine, papaverine and 3-isobutyl-l-methylxanthine (IBMX) were assessed individually, in strips of rabbit renal artery moderately contracted with noradrenaline (NA) in the absence or presence of phosphodiesterase inhibitors (papaverine and IBMX) or verapamil, a Ca(2+) antagonist.2 The vasodilator effect of histamine was potentiated by papaverine (6.1 x 10(-7) M) and IBMX (4.4 x 10(-5) M) but inhibited dose-dependently by verapamil (5.1 and 51.0 x 10(-7) M).3 Adenosine-induced vascular relaxations were greatly increased in the presence of papaverine (6.1 x 10(-7) M) but significantly reduced in the presence of IBMX (4.4 x 10(-5) M) or verapamil (5.1 and 51.0 x 10(-7) M).4 The vasodilatation produced by isoprenaline was increased in the presence of IBMX (4.4 x 10(-5) M) or papaverine (6.1 x 10(-7) M), but inhibited by verapamil (5.1 and 51.0 x 10(-7) M).5 The vascular relaxant effects of nitroglycerine and papaverine were inhibited in the presence of IBMX (4.4 x 10(-5) M) or verapamil (5.1 and 51.0 x 10(-7) M). Papaverine (6.1 x 10(-7) M) also antagonized nitroglycerine-induced vascular relaxation.6 The vasodilator effect of IBMX was greatly reduced in the presence of papaverine (6.1 x 10(-7) M) or verapamil (5.1 and 51.0 x 10(-7) M).7 The vascular relaxant effect of verapamil was reduced proportionally by raising the extracellular Ca(2+) concentration from 1.25 to 5.0 mM while those elicited by histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX were not modified by this procedure.8 These results were taken as an indication that several vasodilators (e.g. histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX), but not a Ca(2+) antagonist such as verapamil, produce a fraction of their vasodilator effects by promoting Ca(2+) extrusion from and/or Ca(2+) sequestration into the vascular smooth muscle cells, via a cyclic adenosine 3',5'-monophosphate-dependent mechanism.

Studies on the mechanism of action of glucagon in strips of rabbit renal artery.

1 The vasodilator effects of glucagon and adenosine cyclic 3',5'-monophosphate (cyclic AMP) were evaluated in strips of rabbit renal artery contracted with noradrenaline (NA) in the absence and presence of phosphodiesterase inhibitors or calcium (Ca(2+)) antagonists.2 The vascular relaxant effect of glucagon was markedly potentiated by various concentrations of four different phosphodiesterase inhibitors (papaverine, theophylline, 3-isobutyl-l-methylxanthine (IBMX) and indomethacin), while that of cyclic AMP was potentiated by only two of them (papaverine and indomethacin) and inhibited by the others (theophylline and IBMX).3 Amongst the four phosphodiesterase inhibitors, IBMX (10 mug/ml) was found to produce the largest potentiation (e.g. the sensitivity increased by a factor of 10) of glucagon-induced vascular relaxations (ED(50) of glucagon in the presence of IBMX = 9.2 +/- 1.0 ng/ml).4 Ca(2+) antagonists such as verapamil and SKF 525A produced a dose-dependent inhibition of the vasodilator action of glucagon. Verapamil (2.5 mug/ml) also antagonized cyclic AMP-induced vascular relaxations.5 The vasodilator effect of verapamil was inhibited dose-dependently by raising the concentration of extracellular Ca(2+) from 0.05 to 0.2 g/l (or 1.25 to 5.0 mM) while those elicited by glucagon or cyclic AMP were not influenced, thus suggesting that the latter two drugs do not interfere with Ca(2+) influx.6 Disodium edetate (Na(2)EDTA, 210 to 840 mug/l) produced a dose-dependent vasodilator effect which was attributed to the facilitation of Ca(2+) extrusion from the smooth muscle cells and/or Ca(2+) binding to the cell membrane. The relaxation produced by Na(2)EDTA was significantly blocked by verapamil (10 mug/ml) or SKF 525A (10 mug/ml).7 The results were taken as an indication that glucagon produces at least a fraction of its vasodilator effect by promoting Ca(2+) extrusion from the vascular smooth muscle cells and/or Ca(2+) binding to or sequestration into intracellular sites, presumably via a cyclic AMP-dependent mechanism.