Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family

Mucin-type O-glycosylation is initiated by a large family of UDP- GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc- transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.      

Are there latitudinal and altidudinal Rapoport effects in the geographic ranges of Andean passerine birds?

We analysed the range-sizes of 835 Andean passerine species (including 414 endemics and 421 non-endemics) to test for latitudinal and altitudinal Rapoport effects (LRE and ARE). We tested for positive range-size: latitude/altitude correlations using three different methods: (i) Rohde's mid-point method, (ii) species sorted out by altitude, and (iii) a phylogenetic comparative method (CAIC). Using Rohde's mid-point method, the mean latitudinal extent of species does not follow a Rapoport pattern, but the mean latitudinal occupancy of all passerines and non-endemics do increase with latitude. The latitudinal ranges of endemics sorted out by altitude follow a reverse Rapoport effect, but non-endemics support the pattern. CAIC confirms the latitudinal increase in the occupancy of non-endemics, but regressions have low coefficients of determination. The ARE is supported by the mean altitudinal extent of species, but the trend vanishes when controlling for geometric effects. Low-altitude species occupy about the same proportion of the available altitudinal space as do high-altitude ones. Our analyses suggest that latitude and altitude have low explanatory power for understanding the spatial variation in range-sizes at a continental scale. We show how different patterns can emerge from applying different criteria to the analysis of data.     

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Isolation and Identification of Novel Microcystin-Degrading Bacteria

Of 31 freshwater bacterial isolates screened using the Biolog MT2 assay to determine their metabolism of the microcystin LR, 10 were positive. Phylogenetic analysis (16S rRNA) identified them as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. This is the first report of microcystin degraders that do not belong to the Proteobacteria.A number of studies have reported biological degradation of microcystin in samples from lakes and sediments (3, 4, 12, 15), but only a few bacterial strains with the ability to degrade microcystins have been isolated and characterized (6). Previously identified bacteria belonged to the Proteobacteria, and with the exception of one isolate (Sphingomonas sp. strain CB4), they were all shown to degrade microcystin LR (MC-LR) via the same degradation pathway: formation of linear MC-LR following cleavage at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg bond and hydrolysis at Ala-Leu to yield a tetrapeptide with Adda as the final product detected (1, 10, 11). Genes encoding enzymes involved in this pathway have been characterized and shown to have similarity in all bacterial isolates currently reported to degrade microcystins (2).Recent work indicated a greater diversity of microbes capable of degrading microcystins and nodularin, with the tentative identification of several novel degradation intermediates (5). In the present study, enrichment was used to isolate bacteria from three Scottish water bodies previously shown to contain microflora capable of microcystin degradation (5). The Biolog MT2 assay was used to screen the ability of the isolated bacteria to metabolize MC-LR, since this had previously been shown to be an effective means of demonstrating metabolism of microcystin by Paucibacter toxinivorans (6).The ability to metabolize MC-LR was determined in the Biolog MT screen, with 10 of the bacterial isolates giving a positive result. We subsequently confirmed that they could all degrade MC-LR in batch degradation studies, as evidenced by liquid chromatography-mass spectrometry (LC-MS) analysis. The microcystin-degrading bacteria were identified by using 16S rRNA gene analysis and investigated to determine the presence of mlrA, mlrB, mlrC, and mlrD, the genes previously reported to be involved in the degradation of MC-LR by Sphingomonas sp. strain ACM-3962 (2). We report here isolates identified as Arthrobacter spp., Brevibacterium sp., and Rhodococcus sp. which have the ability to degrade MC-LR, although none of the previously characterized mlr genes were detected.Surface water samples were collected in sterile Pyrex glass bottles on 26 September 2007 from Loch Rescobie (Ordinance Survey grid reference number NO 52505159), Forfar Loch (NO 293458), and the River Carron (NO 877857), Scotland, United Kingdom. Samples were stored at 4°C overnight and filtered as previously described (5). Aliquots from each water sample (2 × 500 ml) were processed and analyzed by high-performance LC to determine the presence of naturally occurring microcystins (13). Enrichment and shake flask die-away kinetics were monitored in triplicate for each water type (50 ml in sterile 100-ml Erlenmeyer flasks). To enrich bacteria with the ability to degrade a range of different microcystins, three microcystins, selected for their differing polarities, and the pentapeptide nodularin were added to each water sample. MC-LR, MC-RR, MC-LF, and nodularin (Enzo Life Sciences, Lausen, Switzerland) were resuspended in a small volume (100 μl) of methanol and diluted with Milli-Q to a total concentration of 0.4 mg ml⁻¹. The toxin cocktail was sterilized (0.2-μm Dynaguard filter; Fisher, United Kingdom) and added to each flask under aseptic conditions to give a final concentration of 1 μg ml⁻¹of each toxin (i.e., 4 μg ml⁻¹ total concentration). All flasks were incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (2 ml) were removed from each flask under sterile conditions every 2 days, transferred into 4-ml glass vials, and frozen (−20°C) immediately. Die-away kinetics were monitored for 14 days. The frozen samples were freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 minutes. The supernatant (100 μl) was removed for LC-MS analysis (5). Sterile controls (3 × 50 ml) were prepared, incubated, and sampled as described above to confirm whether loss of toxin was a result of microbial activity.After 14 days of enrichment, 1 ml of sample was removed aseptically from each flask, namely, the Loch Rescobie (R), Forfar Loch (F), and River Carron (C) samples. Serial dilutions (to 10⁻⁵) were made using Ringer''s solution (Oxoid Ltd., United Kingdom), and 1 ml of each dilution was removed and mixed with 20 to 25 ml of molten LB agar, poured onto sterile petri dishes, and incubated in the dark at 25°C for 5 days. Colonies with differing morphologies were resuspended in liquid LB medium, and pure cultures were obtained by repeated streaking onto LB agar plates. For the Biolog MT2 assay, a loop of each isolated bacterial strain was transferred to 5 ml of liquid LB medium and incubated overnight in the dark at 25°C. The exponentially growing cultures were then washed twice by centrifugation at 1,000 × g for 15 min, the bacterial pellets were resuspended in sterile 0.01 M phosphate-buffered saline, and the cultures were incubated at 25°C for 24 h to deplete residual carbon. The turbidity of all bacterial suspensions was an A₅₉₀ of 0.35. MC-LR was added to Biolog MT2 plates (Technopath, Limerick, Ireland) in triplicate to give final concentrations of 10, 1, 0.1, and 0 μg ml⁻¹. Wells were inoculated with bacterial suspension (150 μl), and plates were incubated at 25°C. Absorbance at 595 nm was recorded by using a Dynex microplate reader (Jencons, Leighton Buzzard, United Kingdom) immediately after inoculation (0 h) and at 3, 6, 15, 18, 24, and 48 h. Metabolism of MC-LR results in the reduction of tetrazolium violet, giving a color reaction that can be quantified spectroscopically (8). Bacterial isolates found by using the Biolog MT screen to metabolize MC-LR were evaluated for their ability to degrade MC-LR. Isolates C1, C3, and C6 (from the River Carron), F3, F7, and F10 (from Forfar Loch), and R1, R4, R6, and R9 (from Loch Rescobie) were grown overnight in LB liquid medium at 25°C. Bacterial isolates were washed and carbon depleted as described above (0.5 ml) and then added to glass universal bottles containing 9 ml of 0.2-μm-filter-sterilized water from their original locations. Aqueous MC-LR (0.5 ml) was added to each bottle under aseptic conditions at a final concentration of 5 μg ml⁻¹. Triplicate samples were prepared for each isolate and incubated at 25°C ± 1°C with shaking at 100 rpm. Aliquots (0.5 ml) were removed at 24-h intervals under sterile conditions, freeze-dried, reconstituted in 200 μl of 50% aqueous methanol, and centrifuged at 15,000 × g for 10 min. The supernatant (100 μl) was removed for LC-MS analysis performed as previously described (6). Experiments with sterile controls were performed for each water sample. Paucibacter toxinivorans DSMZ-16998 (Braunschweig, Germany) was used as a positive control as it has been reported to degrade MC-LR, MC-YR, and nodularin (16).To identify selected isolates, total DNA was extracted from the pellet by using an UltraClean DNA isolation kit (Mo Bio Laboratories, CA). Sequencing was performed with a BigDye Terminator cycle sequencing reaction kit (202 instrument; Applied Biosystems, United Kingdom) using 8F, 1492, and various other internal primers (518R and 1087R) on an automated DNA sequencer (ABI, United Kingdom) (7, 17). The quality of the sequence was checked by using the sequence analysis software (ABI), and the products of the forward and reverse primers were aligned using Kodon (Applied Maths, Saint-Martens-Latem, Belgium). The analyzed sequences were compared to DNA sequences in public databases using the BLAST function of NCBI (http://www.ncbi.nlm.nih.gov). Individual isolates were classified according to their similarity to sequences in the database. DNA sequences of all isolates, along with those of related bacteria and some known microcystin-degrading bacteria, were used to construct a phylogenetic tree using MEGA4 (18). The sequences were first aligned using Clustal W, and then a phylogenetic tree was constructed by performing neighbor-joining tree analysis with 1,000 bootstrap replicates. Each microcystin-degrading isolate was assayed to determine if mlr genes for the degradation of microcystin could be detected. The PCR method used primers specific for mlrA, mlrB, mlrC, and mlrD with conditions as described before (9). A positive control for these genes was used (Sphingopyxis sp. strain LH21).The Biolog MT2 plates, used to screen 31 isolates, were shown to be an effective means of rapidly identifying bacteria with the ability to metabolize MC-LR (Fig. ​(Fig.1).1). Ten isolates which demonstrated respiration in the presence of MC-LR using the Biolog format were subsequently proven to be microcystin-degrading bacteria in batch studies where MC-LR almost or totally disappeared after 3 days of incubation (Table ​(Table1).1). The results of LC-MS analysis indicated that MC-LR disappeared with no obvious biotransformation or intermediate degradation products. This may be because degradation resulted in only very low concentrations of these compounds and MC-LR is readily utilized by the isolates, as evidenced by respiration in the Biolog assay. Employing the Biolog MT plate enabled rapid (approximately 24 h) selection of bacteria in a high-throughput format (96-well plates) using considerably less microcystin or nodularin, i.e., 5 μg per isolate in the Biolog MT plate compared to 300 μg to follow degradation in die-away kinetics as described herein). Furthermore, following degradation by the latter method requires sample processing and high-performance LC analysis, increasing the time and cost. While the Biolog plates have been widely used for community profiling and bacterial identification, they have yet to be fully exploited in biodegradation studies, where they may facilitate rapid, cost-effective screening of many more bacterial isolates for the ability to utilize a wide range of environmental pollutants.Open in a separate windowFIG. 1.Results of Biolog screen for MC-LR metabolism by bacteria isolated from Loch Rescobie (A), Forfar Loch (B), and the River Carron (C) after 24 h of incubation. Control samples (black bars) contained no additional carbon source. MC-LR was added as the carbon source at 0.1 (open bars), 1 (hatched bars), and 10 (shaded bars) μg ml⁻¹. Error bars represent 1 standard deviation (n = 3).

TABLE 1.

Batch degradation of MC-LR by bacterial isolates in source

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Morphological diversity of the red squirrel, <Emphasis Type="Italic">Sciurus vulgaris</Emphasis>, in Ireland

The red squirrel in Britain and Ireland has been described as a separate subspecies, Sciurus vulgaris leucourus, based on bleaching of the tail and ear tufts. However, recent investigations in northern England found this light colour confined to one area, probably due to the rapid spread of introduced continental European red squirrels. This study reports the first detailed survey of tail colour and cranial measurements in the Irish red squirrel population to (1) investigate the distribution of the light colour morph in Ireland and (2) determine whether the Irish red squirrel population is morphologically divergent from populations elsewhere in the species range. The light tail colour was found in 57% of individuals and in all regions, although it was most common in the northwest. The mixture of different colour morphs indicates the Irish population is a mixture of different subspecies, including S. vulgaris leucourus, while the cranial measurements suggest the Irish squirrel may be morphologically divergent from populations elsewhere. Combined, these results support previous suggestions that conservation measures seek to maintain the diversity within the Irish red squirrel population.     

Focus on Water: Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.Bacteria are commonly employed for the purification of municipal and industrial wastewater but until now, established water treatment technologies have not taken advantage of photosynthetic bacteria (i.e. cyanobacteria). The ability of cyanobacterial cultures to grow at high cell densities with minimal nutritional requirements (e.g. sunlight, carbon dioxide, and minerals) opens up many future avenues for sustainable water treatment applications.Water security is an urgent global issue, especially because many regions of the world are experiencing, or are predicted to experience, water shortage conditions: More than one in six people globally are water stressed, in that they do not have access to safe drinking water (United Nations, 2006). Ninety-seven percent of the Earth’s water is in the oceans; consequently, there are many efforts to develop efficient methods for converting saltwater into freshwater. Various processes using synthetic membranes, such as reverse osmosis, are successfully used for large-scale desalination. However, the high energy consumption of these technologies has limited their application predominantly to countries with both relatively limited freshwater resources and high availability of energy, for example, in the form of oil reserves.The development of an innovative, low-energy biological desalination process, using biological membranes of cyanobacteria, would thus be both attractive and pertinent. The core of the proposed biodesalination process (Fig. 1) is a low-salt biological reservoir within seawater that can serve as an ion exchanger. Its development can be separated into several complementary steps. The first step comprises the selection of a cyanobacterial strain that can be grown to high cell densities in seawater with minimal requirement for energy sources other than those that are naturally available. The environmental conditions during growth can be manipulated to enhance natural extrusion of sodium (Na⁺) by cyanobacteria. In the second step, cyanobacterial ion transport mechanisms must be manipulated to generate cells in which sodium export is replaced with intracellular sodium accumulation. This will involve inhibition of endogenous Na⁺ export and expression of synthetic molecular units that facilitate light-driven sodium flux into the cells. A robust control system built from biological switches will be required to achieve precisely timed expression of the salt-accumulating molecular units. The third step consists of engineering efficient separation of the cyanobacterial cells from the desalinated water, using knowledge of physicochemical properties of the cell surface and their natural ability to produce extracellular polymeric substances (EPSs), which aid cell separation while preserving cell integrity. The fourth step integrates the first three steps into a manageable and scalable engineering process. The fifth and final step assesses potential risks and public acceptance issues linked to the new technology.Open in a separate windowFigure 1.Proposed usage of cyanobacterial cultures for water treatment. A, Hypothetical water treatment station. Situated in basins next to the water source, sun-powered cell cultures remove unwanted elements from the water. The clean water is separated from the cells for human uses. The produced biomass is available for other industries. The proposed biodesalination process is based on the following steps. B, Photoautotrophic cells divide to generate high-density cultures. C, The combined cell volume is low in salt as a result of transport proteins in the cell membrane that export sodium using photosynthetically generated energy. D, Through environmental and genetic manipulation, salt export is inhibited and replaced with transport modules that accumulate salt inside the cells. This process is again fueled by light energy. E, Manipulation of cell surface properties separates the salt-enriched cells from the desalinated water.In this review, we outline the state of knowledge and available technology for each of the steps, as well as summarize the current knowledge gaps and technical limitations in employing a large-scale water treatment process using cyanobacteria. Before discussing these issues, we provide some background information on the usage of cyanobacteria in biotechnology and the impact of sodium on cellular functions of cyanobacteria. The example of biodesalination provides a good vehicle to discuss the suitability of photosynthetic bacteria for water treatment more generally. The issues addressed in this review are relevant for a wide range of biotechnological applications of cyanobacteria, including bioremediation and biodegradation as well as the generation of biofuels, natural medicines, or cosmetics.     

Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases

Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.     

Novel therapeutic strategies based on toll-like receptor signaling

The innate immune system is a critical first line of defense against many microbial, fungal and viral pathogens. Toll-like receptors play a central role in innate immunity, recognizing conserved pathogen-associated molecular patterns and generating signals leading to the initiation of an adaptive immune response. Because of their ability to modulate adaptive immunity, Toll-like receptors represent strategic therapeutic targets for diseases that involve inappropriate adaptive immune responses, such as sepsis, autoimmune disorders, cancer and allergy.