A new principle for tight junction modulation based on occludin peptides

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [C-14] mannitol as a paracellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C-14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C-14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The L- and D-diastereomers of C-14-OP90-103 had distinctly different effects. The D-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The L- isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C-14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.

Effects of chilling temperatures on ethylene binding by banana fruit

Banana fruit are highly susceptible to chilling injury during low temperature storage. Experiments were conducted to compare ethylene binding during storage at chilling (3 and 8 degreesC) versus optimum (13 degreesC) temperatures. The skins of fruit stored at 3 and 8 degreesC gradually darkened as storage duration increased. This chilling effect was reflected in increasing membrane permeability as shown by increased relative electrolyte leakage from skin tissue. In contrast, banana fruit stored for 8 days at 13 degreesC showed no chilling injury symptoms. Exposure of banana fruit to the ethylene binding inhibitor 1-methylcyclopropene (1 mul l(-1) 1-MCP) prevented ripening. However, this treatment also enhanced the chilling injury accelerated the occurrence of chilling injury-associated increased membrane permeability. C-14-ethylene release assay showed that ethylene binding by banana fruit stored at low temperature decreased with reduced storage temperature and/or prolonged storage time. Fruit exposed to 1-MCP for 12 h and then stored at 3 or 8 degreesC exhibited lower ethylene binding than those stored at 13 degreesC. Thus, chilling injury of banana fruit stored at low temperature is associated with a decrease in ethylene binding. The ability of tissue to respond to ethylene is evidently reduced, thereby resulting in failure to ripen.

The effect of region of application on absorption of ethanol and hexanol through canine skin

The effect of region of application on the percutaneous penetration of solutes with differing lipophilicity was investigated in canine skin. Skin from the thorax, neck, back, groin, and axilla regions was harvested from Greyhound dogs and placed in Franz-type diffusion cells. Radiolabelled (C-14) ethanol (Log P 0.19) or hexanol (Log P 1.94) was applied to each skin section for a total of 5 h. The permeability coefficient (k(P), cm h(-1)) and residue of alcohol remaining in the skin were significantly (P = 0.001) higher for hexanol compared to ethanol. In contrast, ethanol had a far greater maximum flux (J(max), mol (cm(2))(-1) h(-1)) than hexanol (P = 0.001). A comparison of regional differences shows the k(P) and Jmax for ethanol in the groin was significantly lower (P = 0.035) than the back. The k(P) and Jmax for hexanol were significantly higher (P = 0.001) in the axilla than the other four skin sites. An understanding of factors influencing percutaneous drug movement is important when formulating topical preparations for the dog. (C) 2003 Elsevier Ltd. All rights reserved.

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3-.N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO3-.N mg of MLVSS-1 h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [C-13] methanol to biomark the DNA of the denitrifiers. The extracted [C-13]DNA and [C-12]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [C-13]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [C-12]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [C-14] methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.

Biosynthetic pathways to isocyanides and isothiocyanates; precursor incorporation studies on terpene metabolites in the tropical marine sponges Amphimedon terpenensis and Axinyssa n.sp.

The biosynthetic origins of the isocyanide and isothiocyanate functional groups in the marine sponge metabolites diisocyanoadociane (1), 9-isocyanopupukeanane (10) and 9- isothiocyanatopupukeanane (11) are probed by the use of [C-14]-labelled precursor experiments. Incubation of the sponge Amphimedon terpenensis with [C-14]-labelled thiocyanate resulted in radioactive diisocyanoadociane ( 1) in which the radiolabel is specifically associated with the isocyanide carbons. As expected, cyanide and thiocyanate were confirmed as precursors to the pupukeananes 10 and 11 in the sponge Axinyssa n. sp.; additionally these precursors labelled 2-thiocyanatoneopupukeanane ( 12) in this sponge. To probe whether isocyanide-isothiocyanate interconversions take place at the secondary metabolite level, the advanced precursor bisisothiocyanate 17 was supplied to A. terpenensis, but did not result in significant labelling in the natural product isocyanide 1. In contrast, in the sponge Axinyssa n. sp., feeding of [C-14]-9-isocyanopupukeanane (10) resulted in isolation of radiolabelled 9- isothiocyanatopupukeanane 11, while the feeding of [C-14]-11 resulted in labelled isocyanide 10. These results show conclusively that isocyanides and isothiocyanates are interconverted in the sponge Axinyssa n. sp., and confirm the central role that thiocyanate occupies in the terpene metabolism of this sponge.

Biosynthetic pathways to dichloroimines; precursor incorporation studies on terpene metabolites in the tropical marine sponge Stylotella aurantium

The biosynthetic origin of the dichloroimine functional group in the marine sponge terpene metabolites stylotellanes A ( 3) and B ( 4) was probed by the use of [C-14]-labelled precursor experiments. Incubation of the sponge Stylotella aurantium with [C-14]-labelled cyanide or thiocyanate resulted in radioactive terpenes in which the radiolabel was shown by hydrolytic chemical degradation to be associated specifically with the dichloroimine carbons. Additionally, label from both precursors was incorporated into farnesyl isothiocyanate ( 2). A time course experiment with [ 14C]cyanide revealed that the specific activity for farnesyl isothiocyanate decreases over time, but increases for stylotellane B ( 4), consistent with the rapid formation of farnesyl isothiocyanate ( 2) from inorganic precursors followed by a slower conversion to stylotellane B ( 4). The advanced precursors farnesyl isothiocyanate ( 2) and farnesyl isocyanide ( 5) were supplied to S. aurantium, and shown to be incorporated efficiently into stylotellane A ( 3) and B ( 4). Feeding of [C-14]-farnesyl isothiocyanate ( 2) resulted in a higher incorporation of label than with [C-14]-farnesyl isocyanide ( 5). Farnesyl isocyanide was incorporated into farnesyl isothiocyanate in agreement with labelling studies in other marine sponges. Both farnesyl isocyanide and isothiocyanate were further incorporated into axinyssamide A ( 11) as well as the cyclized dichloroimines (12)-(14), ( 16) that represent more advanced biosynthetic products of this pathway. These results identify the likely biosynthetic pathway leading to the major metabolites of S. aurantium.

Archaeology and age of a new hominin from Flores in eastern Indonesia

Excavations at Liang Bua, a large limestone cave on the island of Flores in eastern Indonesia, have yielded evidence for a population of tiny hominins, sufficiently distinct anatomically to be assigned to a new species, Homo floresiensis(1). The finds comprise the cranial and some post-cranial remains of one individual, as well as a premolar from another individual in older deposits. Here we describe their context, implications and the remaining archaeological uncertainties. Dating by radiocarbon (C-14), luminescence, uranium-series and electron spin resonance (ESR) methods indicates that H. floresiensis existed from before 38,000 years ago (kyr) until at least 18 kyr. Associated deposits contain stone artefacts and animal remains, including Komodo dragon and an endemic, dwarfed species of Stegodon. H. floresiensis originated from an early dispersal of Homo erectus ( including specimens referred to as Homo ergaster and Homo georgicus)(1) that reached Flores, and then survived on this island refuge until relatively recently. It overlapped significantly in time with Homo sapiens in the region(2,3), but we do not know if or how the two species interacted.

Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia

Large blooms of the marine cyanobacterium Lyngbya majuscula in Moreton Bay, Australia (27 degrees 05'S, 153 degrees 08'E) have been re-occurring for several years. A bloom was studied in Deception Bay (Northern Moreton Bay) in detail over the period January-March 2000. In situ data loggers and field sampling characterised various environmental parameters before and during the L. majuscula bloom. Various ecophysiological experiments were conducted on L. majuscula collected in the field and transported to the laboratory, including short-term (2h) C-14 incorporation rates and long-term (7 days) pulse amplitude modulated (PAM) fluorometry assessments of photosynthetic capacity. The effects of L. majuscula on various seagrasses in the bloom region were also assessed with repeated biomass sampling. The bloom commenced in January 2000 following usual December rainfall events, water temperatures in excess of 24 degrees C and high light conditions. This bloom expanded rapidly from 0 to a maximum extent of 8 km(2) over 55 days with an average biomass of 210 g(dw)(-1) m(-2) in late February, followed by a rapid decline in early April. Seagrass biomass, especially Syringodium isoetifolium, was found to decline in areas of dense L. majuscula accumulation. Dissolved and total nutrient concentrations did not differ significantly (P > 0.05) preceding or during the bloom. However, water samples from creeks discharging into the study region indicated elevated concentrations of total iron (2.7-80.6 mu M) and dissolved organic carbon (2.5-24.7 mg L-1), associated with low pH values (3.8-6.7). C-14 incorporation rates by L. majuscula were significantly (P < 0.05) elevated by additions of iron (5 mu M Fe), an organic chelator, ethylenediaminetetra-acetic acid (5 mu M EDTA) and phosphorus (5 mu M PO4-3). Photosynthetic capacity measured with PAM fluorometry was also stimulated by various nutrient additions, but not significantly (P > 0.05). These results suggest that the L. majuscula bloom may have been stimulated by bioavailable iron, perhaps complexed by dissolved organic carbon. The rapid bloom expansion observed may then have been sustained by additional inputs of nutrients (N and P) and iron through sediment efflux, stimulated by redox changes due to decomposing L. majuscula mats. (c) 2004 Elsevier B.V. All rights reserved.

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [C-13]acetate was used in SIP to label the DNA of the denitrifiers. The [C-13]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the C-13 library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking Up [C-14] acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the waste-water industry to enhance denitrification.

Effects of vehicle and region of application on absorption of hydrocortisone through canine skin

Objective-To determine the effects of various vehicles on the penetration and retention of hydrocortisone applied to canine skin. Sample Population-20 canine skin samples obtained from the thorax, neck, and groin regions of 5 Greyhounds. Procedure-Skin was harvested from dogs after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of hydrocortisone that contained trace amounts of radiolabelled [C-14]-hydrocortisone in each vehicle (ie, PBS solution [PBSS] alone, 50% ethanol [EtOH] in PBSS [wt/wt], and 50% propylene glycol in PBSS [wt/wt]) were applied to the outer (stratum corneum) surface of each skin sample, and aliquots of receptor fluid were collected for 24 hours and analyzed for hydrocortisone. Results-The maximum flux of hydrocortisone was significantly higher for all sites when dissolved in a vehicle containing 50% EtOH, compared with PBSS alone or 50% propylene glycol, with differences more prominent in skin from the neck region. In contrast, higher residues of hydrocortisone were found remaining within the skin when PBSS alone was used as a vehicle, particularly in skin from the thorax and neck. Conclusions and Clinical Relevance-Penetration of topically applied hydrocortisone is enhanced when EtOH is used in vehicle formulation. Significant regional differences (ie, among the thorax, neck, and groin areas) are also found in the transdermal penetration and skin retention of hydrocortisone. Variability in clinical response to hydrocortisone can be expected in relation to formulation design and site of application.

Radiocarbon variations from the southern hemisphere, 10,350-9700 cal BP

We have made AMS measurements on a series of 10-ring samples from a subfossil Huon pine log found in western Tasmania (42degreesS, 145degreesE). The results show a pronounced rise in Delta(14)C over the first 200 years, and a decrease over the following 160 years. Tree-ring width measurements indicate that this log (catalogue SRT-447) can be cross-dated with another subfossil log (SRT-416) for which a series of high-precision radiometric C-14 measurements have previously been made. When the two tree-ring series are thus aligned, SRT-447 is the older of the two logs, and there is a 139-year overlap. We then have a Huon pine floating chronology spanning 680 years, with C-14 measurements attached. The C-14 data sets agree well within the period of overlap indicated by the tree-rings. The C-14 variations from Huon pine show excellent agreement with those from German oak and pine for the period 10,350-9670 cal BP. Aligning the Huon pine C-14 Series with that from German oak and pine allows us to examine the inter-hemispheric offset in C-14 dates in the early Holocene. (C) 2004 Elsevier B.V. All rights reserved.

Thorium-230 coral chronology of a late prehistoric Hawaiian chiefdom

Documenting the history of settlement in Hawaii during the last few Centuries before European contact, is crucial to charting the evolution of the most complex chiefdom in Polynesia. It is precisely this period that Hawaii. and many Polynesian Societies, Underwent their most rapid changes in political, economic and social organisation. The last similar to 500 to 300 years in the C-14 calibration curve is problematic with wide fluctuations Often rendering large age spans that do not precisely date single events, especially troubling with a culture-historical record of similar to 1000 years duration. Here we present in extremely high precision Th-230 chronology for archaeologically constrained coral samples from a range of occupancy sites. Our high precision dates allow the time of site use to be clearly demonstrated. They also provide the first dates for habitation sites in Hawaii that clearly show contemporaneous occupation-the major problem in settlement pattern archaeology. We demonstrate that two sites were occupied within the same year. Our refined chronology, provides new and exciting oppurtunities for tracking sociopolitical and economic developments during the last few centuries-the crucial period in the evolution and transformation of Polynesian societies. (c) 2005 Elsevier Ltd. All rights reserved.

Intra-colonial response to Acroporid "White syndrome" lesions in tabular Acropora spp. (Scleractinia)

'White syndrome' is considered to be the most prevalent coral disease on the Great Barrier Reef, characterised by rapid rates of lesion progression and high levels of colony mortality. This study investigated the production and translocation of photoassimilates towards white syndrome lesions (WSLs) and artificially inflicted lesions in healthy and diseased colonies of tabular Acropora spp. to determine the intra-colonial response to white syndrome using C-14 labelling. Translocation of C-14 labelled photoassimilates was preferentially orientated away from active WSLs, with minimal C-14 activity observed in the lesion borders, whilst artificial lesions (ALs) created directly opposite WSL borders showed significantly higher C-14 activity, suggesting active translocation of photoassimilates for tissue regeneration. Transport of photoassimilates in healthy coral colonies was preferentially oriented towards ALs with a higher perimeter-area ratio, although translocation towards WSL boundaries was minimal even though the lesion perimeter was often the width of the colony (> 200 cm). We suggest that the preferential orientation of photoassimilates away from WSLs may represent a deliberate strategy by the colony to induce a 'shutdown reaction' in order to preserve intra-colonial resources within areas of the colony that are more likely to survive and recover.

Relationship between bicarbonate retention and bone characteristics in broiler chickens

Determination of the bicarbonate retention factor (BRF) is an important step during development of the indicator amino acid oxidation technique for use in a new model. A series of 4-h oxidation experiments were performed to determine the BRF of broilers aged 7, 14, 21, 28, 35, and 42 d using 4 birds per age group. A priming dose of 1.2 mu Ci of (NaHCO3)-C-14, followed by eight half-hourly doses of 1 mu Ci of (NaHCO3)-C-14 were given orally to each of 4 birds per age. The percentage of 14 C dose expired by the bird at a steady state was measured. These birds, as well as 12 additional birds matched for age and BW, were killed, and femur bone mineral density was measured by quantitative computed tomography to determine the relationship between bone development and bicarbonate retention at each age. There was a correlation (r = 0.50; P < 0.05) between total cross-sectional femur bone mineral density and bicarbonate retention at each age. A prediction equation (Y = 6.95 x 10(-2) X - 3.51 x 10(5)X(2) + 27.58; P < 0.0001, R-2 = 0.79) where Y = bicarbonate retention and X = BW was generated to predict Y as a function of X. Bicarbonate retention values peaked at 28 d, during the stage of the most rapid bone deposition and the highest growth rate. A constant BRF was found from 1,900 to 2,700 g of BW of 35.15 +/- 1.095% (mean SEM). This retention factor will allow the accurate correction of oxidation of C-14-labeled substrates in broilers of different ages and BW in future indicator amino acid oxidation studies.