Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Determination of In Situ Bacterial Growth Rates in Aquifers and Aquifer Sediments

Laboratory and field-scale studies with stained cells were performed to monitor cell growth in groundwater systems. During cell division, the fluorescence intensity of the protein stain 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) for each cell is halved, and the intensity can be tracked with a flow cytometer. Two strains of bacteria, Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107, both isolated from a shallow aquifer, were utilized in this study. The change in the average generation or the average fluorescence intensity of the CFDA/SE-stained cells could be used to obtain estimates of doubling times. In microcosm experiments, the CFDA/SE-based doubling times were similar to the values calculated by total cell counting and were independent of cell concentration. Intact and repacked sediment core experiments with the same bacteria indicated that changes in groundwater chemistry were just as important as growth rates in determining planktonic cell concentrations. The growth rates within the sediment cores were similar to those calculated in microcosm experiments, and preferential transport of the daughter cells was not observed. The experiments indicated that the growth rates could be determined in systems with cell losses due to other phenomena, such as attachment to sediment or predation. Application of this growth rate estimation method to data from a field-scale bacterial transport experiment indicated that the doubling time was approximately 15 days, which is the first known direct determination of an in situ growth rate for bacteria in an aquifer.     

Measurement of Denitrification in Two Freshwater Sediments by an In Situ Acetylene Inhibition Method

An acetylene inhibition method was satisfactorily used for the in situ measurement of denitrification in two sediment-water systems incubated for not more than 22 h. In the presence of added nitrate, denitrification acted as a source of nitrous oxide in a drainage pond, but acted as a sink in its absence. The averaged rates of nitrous oxide accumulation with nitrate enrichment in the absence and presence of acetylene were 0.15 and 0.30 mg of N m⁻²h⁻¹, respectively. Acetylene reduction at an average rate of 0.07 mmol of C₂H₄ formed m⁻²h⁻¹ was simultaneously measured in the absence of added nitrate. In a small eutrophic lake where nitrogen was nonlimiting, the in situ rates of sediment denitrification were 0.09 and 0.11 mg of N m⁻²h⁻¹ in the presence and absence of macrophytes, respectively, and no acetylene reduction activity was found.     

Comparison of In Vitro Release Rates of Acyclovir from Cream Formulations Using Vertical Diffusion Cells

Acyclovir, indicated in the treatment of herpes labialis (“cold sores”), is formulated as semisolid topical dosage forms and marketed in numerous countries. Since the formulations of the various acyclovir products may differ from country to country, this study was undertaken to compare the in vitro release of acyclovir from various generic cream products available on the South African and Indian markets using the respective brand/innovator product as the reference product. The in vitro studies were carried out using vertical diffusion cells with a diffusional surface area of 1.767 cm² and various commercially available membranes. Normal saline was used as receptor fluid and the temperature maintained at 32 ± 0.5°C. The in vitro release comparisons were based on the recommendations described in the US Food and Drug Administration Draft Guidance for acyclovir ointment and the SUPAC-SS Guidance for non-sterile semisolid dosage forms. The release rates (slope) of the test (T) and the relevant reference product (R) were monitored and compared. The comparative release of acyclovir from the various generic formulations compared with the reference product was found to be within the limits of 75–133.33% with a 90% confidence interval. These experiments indicate that the generic acyclovir cream formulations exhibited release rates that were comparable to the innovator product and could be considered to be bioequivalent.KEY WORDS: acyclovir cream, FDA Guidance, in vitro release, membranes, vertical diffusion cells     

Seasonal Rates of Methane Oxidation in Anoxic Marine Sediments

Methane concentrations and rates of methane oxidation were measured in intact sediment cores from an inshore marine sediment at Jutland, Denmark. The rates of methane oxidation, determined by the appearance of ¹⁴CO₂ from injected ¹⁴CH₄, varied with sediment depth and season. Most methane oxidation was anoxic, but oxygen may have contributed to methane oxidation at the sediment surface. Cumulative rates (0- to 12-cm depth) for methane oxidation at Kysing Fjord were 3.34, 3.48, 8.60, and 17.04 μmol m⁻² day⁻¹ for April (4°C), May (13°C), July (17°C), and August (21°C), respectively. If all of the methane was oxidized by sulfate, it would account for only 0.01 to 0.06% of the sulfate reduction. The data indicate that methane was produced, in addition to being oxidized, in the 0- to 18-cm sediment stratum.     

Biodegradable Injectable In Situ Implants and Microparticles for Sustained Release of Montelukast: In Vitro Release,Pharmacokinetics, and Stability

The objective of this study was to investigate the sustained release of a hydrophilic drug, montelukast (MK), from two biodegradable polymeric drug delivery systems, in situ implant (ISI) and in situ microparticles (ISM). N-Methyl pyrrolidone (NMP), dimethyl sulfoxide (DMSO), triacetin, and ethyl acetate were selected as solvents. The release of 10% (w/v) MK from both systems containing poly-lactic-co-glycolic acid (PLGA) as the biodegradable polymer was compared. Upon contact with the aqueous medium, the PLGA in ISI and ISM systems solidified resulting in implants and microparticles, respectively. The in vitro drug release from the ISI system showed marked difference from miscible solvents (NMP and DMSO) than the partially miscible ones (triacetin and ethyl acetate), and the drug release decreased with increased PLGA concentration. In the ISM system, the initial in vitro drug release decreased with decreased ratio of polymer phase to external oil phase. In vivo studies in rats showed that ISM had slower drug release than the drug release from ISI. Also, the ISM system when compared to ISI system had significantly reduced initial burst effect. In vitro as well as the in vivo studies for both ISI and ISM systems showed sustained release of MK. The ISM system is suitable for sustained release of MK over 4-week period with a lower initial burst compared to the ISI system. Stability studies of the ISI and ISM formulations showed that MK is stable in the formulations stored at 4°C for more than 2 years.     

Studies on Mefenamic Acid Microparticles: Formulation, In Vitro Release, and In Situ Studies in Rats

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.     

In Situ Reproductive Rate of Freshwater Caulobacter spp.

Electron microscope grids were submerged in Lake Washington, Seattle, Wash., in June 1996 as bait to which Caulobacter sp. swarmers would attach and on which they would then reproduce in situ. Enumeration of bands in the stalks of attached cells implied that the caulobacters were completing approximately three reproductive cycles per day. A succession of morphological types of caulobacters occurred, as well as an episode of bacteriovore grazing that slowed the accumulation of caulobacters and prevented the aging of the population.     

In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

New rRNA-targeting oligonucleotide probes permitted the fluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora acuminata), TRIang322 (Tricladium angulatum), and Alongi340 (Anguillospora longissima), are targeted against 18S rRNA, whereas probes TmarchB10, TmarchC1_1, TmarchC1_2, and AlongiB16 are targeted against the 28S rRNA of Tetracladium marchalianum and Anguillospora longissima, respectively. After 2 weeks and 3 months of exposure of polyethylene slides in the stream, attached germinating conidia and growing hyphae of freshwater fungi were accessible for FISH. Growing hyphae and germinating conidia on leaves and in membrane cages were also visualized by the new FISH probes.     

In Vitro Reconstitution of Neurotransmitter Release

The vesicular hypothesis has stimulated fruitful investigations on many secreting systems. In the case of rapid synaptic transmission, however, the hypothesis has been found difficult to reconcile with a number of well established observations. Brief impulses of transmitter molecules (quanta) are emitted from nerve terminals at the arrival of an action potential by a mechanism which is under the control of multiple regulations. It is therefore not surprising that quantal release could be disrupted by experimental manipulation of a variety of cellular processes, such as a) transmitter uptake, synthesis, or transport, b) energy supply, c) calcium entry, sequestration and extrusion, d) exo- or endocytosis, e) expression of vesicular and plasmalemmal proteins, f) modulatory systems and second messengers, g) cytoskeleton integrity, etc. Hence, the approaches by ablation strategy do not provide unequivocal information on the final step of the release process since there are so many ways to stop the release. We propose an alternate approach: the reconstitution strategy. To this end, we developed several preparations for determining the minimal system supporting Ca²⁺-dependent transmitter release. Release was reconstituted in proteoliposomes, Xenopus oocytes and transfected cell lines. Using these systems, it appears that a presynaptic plasmalemmal proteolipid, that we called mediatophore should be considered as a key molecule for the generation of transmitter quanta in natural synapses.     

Comparison of the Long-Term Risks of Removal and In Situ Management of Contaminated Sediments in the Fox River

The long-term potential risks of environmental dredging vs. in situ contaminated sediment management practices are discussed and compared for the Lower Fox River, Wisconsin. The risks are identified as being largely associated with the residual sediment contamination associated with either approach. The integral of the surface area-weighted average contaminant concentration in surface sediment is proposed as a metric to compare these risks. Capping is shown to exhibit significantly reduced exposure and risk relative to the dredging scenarios, even if potential undetected erosion of 5% of the cap is considered. Even with the improbable event of undetected failure of 25% of the cap, the exposure and risk associated with capping is approximately equal to or below all dredging scenarios. A preference for dredging due to the perception that it eliminates the long-term risk of in situ capping is not supported by this analysis. Although strictly applicable only to the Lower Fox River, the results suggest sitespecific analyses must be conducted to determine which sediment management approaches minimize the potential for long-term exposure and risk.     

In Vitro and In Situ Absorption of Methionine Sulfoxide in Rat Small Intestine

Absorption of methionine and its sulfoxide was investigated in vitro with everted sacs and in situ with circulated loops of rat small intestine. Transmural transport and tissue accumulation of methionine sulfoxide in the everted sacs were in fair agreement with those of methionine. Apparent kinetic parameters for the difference of transmural transport in the absence and presence of 10^?5 m carbonylcyanide m-chlorophenylhydrazone, i.e. for the energy-dependent active transport, were similar for both methionine and its sulfoxide. Methionine was found at a low level in the serosal fluid of the everted sac on incubation with methionine sulfoxide. It was attributed to the methionine leaked out from the tissue but not to that formed by reduction of methionine sulfoxide during the course of intestinal transport. Similar transport was also observed in situ in circulated intestinal loops for methionine and its sulfoxide. The absorption efficiency of methionine sulfoxide in the small intestine is not the reason for the decreased nutritional availability of the most likely oxidation product of methionine.     

Cable Bacteria in Freshwater Sediments

In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures and electric fields indicated electron transfer between vertically separated anodic and cathodic half-reactions. Fluorescence in situ hybridization revealed the presence of Desulfobulbaceae filaments. In addition, in situ measurements of oxygen, pH, and electric potential distributions in the waterlogged banks of Giber Å demonstrated the presence of distant electric redox coupling in naturally occurring freshwater sediment. At the same site, filamentous Desulfobulbaceae with cable bacterium morphology were found to be present. Their 16S rRNA gene sequence placed them as a distinct sister group to the known marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary origin of the cable phenotype within Desulfobulbaceae with subsequent diversification into a freshwater and a marine lineage.     

In Situ and In Vitro Expression of Protein Kinase C Alpha in Human Melanocytes

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-α, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-α in frozen neonatal human foreskin exhibited intermittent 2–3+ staining along the basal cell layer consistent with melanocytes, and 0–1+ staining of keratinocytes (on a scale of 0–3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-α and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-α showed strong PKC-α mRNA expression in cultured melanocytes, whereas PKC-α mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-α band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-α mRNA and approximately 6-fold more PKC-α protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-α provides further evidence for cell type specificity in the balance of PKC-α expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.     

Estimation of Sediment Denitrification Rates at In Situ Nitrate Concentrations

The denitrification rates in a marine sediment, estimated by using ¹⁵N-nitrate, V_max, K_m, and sediment nitrate concentrations, were 12.5 and 2.0 nmol of N₂-N cm⁻³ day⁻¹ at 0 to 1 and 1 to 3 cm, respectively, at 12°C. The total rate was 165 nmol of N₂-N m⁻² day⁻¹.     

In Vitro Studies of Ferritin Iron Release and Neurotoxicity

Abstract: The increase in brain iron associated with several neurodegenerative diseases may lead to an increased production of free radicals via the Fenton reaction. Intracellular iron is usually tightly regulated, being bound by ferritin in an insoluble ferrihydrite core. The neurotoxin 6-hydroxydopamine (6-OHDA) releases iron from the ferritin core by reducing it to the ferrous form. Iron release induced by 6-OHDA and structurally related compounds and two other dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium iodide (MPP⁺) and 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), were compared, to identify the structural characteristics important for such release. 1,2,4-Trihydroxybenzene (THB) was most effective in releasing ferritin-bound iron, followed by 6-OHDA, dopamine, catechol, and hydroquinone. Resorcinol, MPP⁺, and TaClo were ineffective. The ability to release iron was associated with a low oxidation potential. It is proposed that a low oxidation potential and an ortho -dihydroxyphenyl structure are important in the mechanism by which ferritin iron is mobilized. In the presence of ferritin, both 6-OHDA and THB strongly stimulated lipid peroxidation, an effect abolished by the addition of the iron chelator deferoxamine. These results suggest that ferritin iron release contributes to free radical-induced cell damage in vivo.     

Nitrification in Freshwater Sediments as Influenced by Insect Larvae: Quantification by Microsensors and Fluorescence in Situ Hybridization

Sediment-reworking macrofauna can stimulate nitrification by increasing the O₂ penetration into sediments or it can reduce nitrification by grazing on nitrifying bacteria. We investigated the influence of Chironomus riparius larvae (Insecta: Diptera) on the in situ activity, abundance, and distribution of NH₄⁺-oxidizing (AOB) and NO₂^–-oxidizing bacteria (NOB) in two freshwater sediments with microsensors and fluorescence in situ hybridization. In organic-poor sediment, nitrification activity was reduced by the presence of C. riparius larvae, whereas no such effect was detected in organic-rich sediment. We explain this difference with the variable larval burrowing and grazing behavior in the two sediment types: In organic-poor sediment larval activities were intense and evenly distributed across the whole sediment surface, whereas in organic-rich sediment larval activities were locally restricted to the microenvironment of animal burrows. Surprisingly, the animals did not cause any significant change of the abundance of AOB and NOB. This implies that the observed reduction of nitrification activity was not density-regulated, but rather was due to the lowered metabolic activity of the nitrifiers. Partial digestion and redeposition of particle-associated bacteria by C. riparius larvae are believed to have caused this loss of metabolic activity.This revised version was published online in November 2004 with corrections to Volume 48.     

Comparison ofin Situ andin Vitro survival ofCandida albicans in seawater

The survival in seawater of several laboratory and field isolates ofCandida albicans was investigated. Initial studies were madein vitro (flasks) to confirm previous reports. Frequent sampling of viable cells showed that flask experiments, even repeated, produced varied patterns of survival in this closed system. As an alternative, multiple experiments were run in untreated seawater in dialysis bags and plexiglas chambers at ambient temperature (17 to 22C) in flowing seawater. Die-off rates of all cultures tested in dialysis bags were very rapid in the first day and may have been related to high levels of dissolved organic carbon in the tubing. Distilled water-or acid-washed bags did not yield significantly higher survival rates in all cases. When plexiglas chambers closed with Nuclepore membranes were used, survival rates decreased to 5% to 15% of the original population after 6 days. Chamber data were more uniform and represented approximately a twofold increase in survival over that shown previously inin vitro (flask) studies. Some evidence was obtained in all three test systems for the greater survival rate of a field isolate ofC. albicans compared with that noted for a laboratory (ATCC) strain. The results are considered to more accurately depict the survival ofC. albicans in summer temperate recreational waters.