Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.     

Combinatorial evolution of high-affinity peptides that bind to the Thomsen-Friedenreich carcinoma antigen

Thomsen-Friedenreich (TF) antigen occurs on approximately 90% of human carcinomas, is likely involved in carcinoma cell homotypic aggregation, and has clinical value as a prognostic indicator and marker of metastasized cells. Previously, we isolated anti-TF antigen peptides from bacteriophage display libraries. These bound to TF antigen on carcinoma cells but were of low affinity and solubility. We hypothesized that peptide amino acid sequence changes would result in increased affinity and solubility, which would translate into improved carcinoma cell binding and increased inhibition of aggregation. The new peptides were more soluble and exhibited up to fivefold increase in affinity (K_d 60 nM). They bound cultured human breast and prostate carcinoma cells at low concentrations, whereas the earlier peptides did not. Moreover, the new peptides were potent inhibitors of homotypic aggregation. The maturated peptides will have expanded applications in basic studies of the TF antigen and particular utility as clinical carcinoma-targeting agents.     

Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 g/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.     

Synthesis and visualization of a membrane-permeable MRI contrast agent

The study of in vivo developmental events has undergone significant advances with the advent of biological molecular imaging techniques such as computer enhanced light microscopy imaging, positron emission tomography (PET), micro-CT, and magnetic resonance imaging (MRI). MRI has proven to be a particularly powerful tool in clinical and biological settings. Images can be acquired of opaque living animals, with the benefit of tracking events of extended periods of time on the same specimen. Contrast agents are routinely used to enhance regions, tissues, and cells that are magnetically similar but histologically distinct. A principal barrier to the development of MR contrast agents for investigating developmental biological questions is the ability to deliver the agent across cellular membranes. As part of our research, we are investigating a number of small molecules that facilitate transport of charged and uncharged species across cell membranes. Here we describe the synthesis and testing of a Gd(III)-based MR contrast agent conjugated to polyarginine that is able to permeate cell membranes. We confirmed cellular uptake of the agent using two-photon laser microscopy to visualize a Eu(III) derivative of the contrast agent in cell culture, and verified this uptake by T₁ analysis of the Gd(III) agent in cells.Abbreviations DOTA 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid - DOTA(tris-t-Bu ester) 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid-tert-butyl ester)-10-acetic acid - DO3A(tris-t-Bu ester) 1,4,7-tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane - MRI magnetic resonance imaging - PET positron emission tomography - TPLM two-photon laser microscopy     

Microstructural characteristics of thin biofilms through optical and scanning electron microscopy

The combination of a conventional optical microscope with a specially designed glass flow cell was used to visualize in situ biofilms formed on opaque thin biomaterials through a simple non-invasive way (optical microscopy of thin biofilms, OMTB). Comparisons of OMTB with scanning electron microscopy (SEM) images were made. Thin metallic dental biomaterials were used as substrata. They were immersed in a synthetic saliva and in a modified Mitis–Salivarius medium inoculated with a consortium of oral microorganisms. To study the effect of bacterial motility, Pseudomonas fluorescens cultures were also used. The processes which give rise to the formation of the biofilm were monitored through OMTB. Biofilm microstructures like pores, water channels, streamers and chains of Streptococci, attached to the surface or floating in the viscous interfacial environment, could be distinguished. Thickness and roughness of the biofilms formed on thin substrata could also be evaluated. Distortions introduced by pretreatments carried out to prepare biological materials for SEM observations could be detected by comparing OMTB and SEM images. SEM images (obtained at high magnification but ex situ, not in real time and with pretreatment of the samples) and OMTB images (obtained in situ, without pretreatments, in real time but at low magnification) in combination provided complementary information to study biofilm processes on thin substrata.     

Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy

The green fluorescent protein (GFP) and other intrinsically fluorescent proteins (IFPs) are popular reporters because they allow visualization of cellular constituents in living specimens. IFP technology makes it possible to view dynamic processes in living cells, but extended observation, using fluorescence microscopy (both wide-field and confocal), can result in significant light energy exposure. Therefore, it is possible that cells experience light-induced damage that alters cell physiology and confounds observations. To understand the impact that extended viewing has on cells, we obtained quantitative information about the effect of light energy dose and observation conditions on tobacco BY-2 cell physiology. Our results show a non-linear relationship between the excitation light intensity and mitotic arrest, and the frequency of mitotic arrest is dependent on the presence of an IFP that absorbs the excitation light. Moreover, fluorescence microscopy induces the production of reactive oxygen species (ROS), as assayed using BY-2 cells loaded with oxidation-sensitive dyes, and the level of ROS production increases if the cells express an IFP that absorbs the excitation light energy. The dye oxidation follows sigmoidal kinetics and is reversible if the cells are exposed to low irradiation levels. In addition, the dye oxidation rate shows a non-linear relationship to the excitation light intensity, and a good correlation exists between photobleaching, mitotic arrest, and dye oxidation. The data highlight the importance of ROS scavenging for normal mitotic progression, and provide a reference for judiciously choosing conditions that avoid photobleaching that can lead to ROS accumulation and physiological damage.     

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).     

Supramolecular dynamics of gap junctions

During the life cycle of a membrane protein its molecular structure may change and for aggregated proteins this process may be observed on the supramolecular level. Here we demonstrate that this is the case for gap junction channels which maintain cell-cell communication. Freshly synthesized connexins are integrated as hexamers (connexons) into the plasma membrane where they form plaques after pairing with connexons of an attached cell. We inhibited protein trafficking by applying the fungal metabolite brefeldin A (BFA), quantified cell-cell coupling by calcein transfer and fluorescence-activated flow cytometry, and examined the degradation and formation of gap junction plaques by indirect immunofluorescence and immunogold labeling. Under control conditions 50% of the detected plaques were ubiquitylated and less than 10% showed a two-dimensional crystalline packing. One hour after BFA reversal about 60% of the plaques were crystalline and ubiquitylation dropped to 14%. Label for ubiquitin was predominantly found on non-crystalline plaques. We, therefore, conclude that newly formed gap junction plaques are of crystalline morphology which changes to a pleomorphic structure when individual channels are modified during their aging process. This dynamic in plaque morphology correlates with channel inactivation and plaque ubiquitylation.     

Myelin basic protein component C1 in increasing concentrations can elicit fusion, aggregation, and fragmentation of myelin-like membranes

Myelin basic protein (MBP) is considered to have a primary role in the formation and maintenance of the myelin sheath. Many studies using artificial vesicle systems of simple lipid composition, and generally small size, have shown that MBP can elicit vesicle fusion, aggregation, or even fragmentation under different conditions. Here, we have studied the effects of increasing concentrations of bovine MBP charge isomer C1 (MBP/C1) on large unilamellar vesicles (LUVs) composed of phosphatidylcholine and phosphatidylserine (92:8 molar ratio), or with a lipid composition similar to that of the myelin membrane in vivo (Cyt-LUVs). Using absorbance spectrophotometry, fluorescence resonance energy transfer, dynamic light scattering and transmission electron microscopy, we have shown that vesicle aggregation and some vesicle fusion occurred upon addition of MBP/C1, and as the molar protein-lipid ratio increased. Fragmentation of Cyt-LUVs was observed at very high protein concentrations. These results showed that the phenomena of vesicle fusion, aggregation, and fragmentation can all be observed in one in vitro system, but were dependent on lipid composition and on the relative proportions of protein and lipid.     

Tracking chromaffin granules on their way through the actin cortex

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a ∼300-nm (1/e²) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D ⁽³⁾). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D ⁽³⁾ was about 10,000 times lower than expected for free diffusion. Granules located ∼60 nm beneath the plasma membrane moved on random tracks (D ⁽³⁾≈10⁻¹⁰ cm² s⁻¹) with several reversals in direction before they approached their docking site at angles larger than 45^∘. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D ⁽³⁾ of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex. Received: 18 November 1999 / Revised version: 26 January 2000 / Accepted: 2 February 2000