Ecological and biogeographic relationships of class Flavobacteria in the Southern Ocean

The abundance, spatial distribution and diversity of class Flavobacteria were investigated in the Southern Ocean euphotic zone across a latitudinal transect and in the ice pack off Eastern Antarctica. Surface seawater samples filter-fractionated into 0.8 mum particulate and 0.2 m planktonic fractions were investigated with different molecular techniques. The abundance of particle-associated Flavobacteria, ascertained with real-time PCR and DGGE band analysis using Flavobacteria-specific primers, was found to be significantly higher in Polar Front Zone (PFZ) and Antarctic Zone (AZ) water samples than in nutrient limited Temperate Zone (TZ) and Sub-Antarctic Zone (SAZ) waters. Abundance of particle-associated Flavobacteria correlated positively with seawater chlorophyll a and nutrient concentrations, suggesting that increased Flavobacteria abundance may relate to enhanced primary production in the PFZ and AZ. This is supported by comparison of DGGE profiles that demonstrated significant differences in the total Flavobacteria community structure and 16S rRNA gene diversity between samples from the PFZ and AZ and those from TZ and SAZ. Sequence analysis revealed a broad diversity amongst class Flavobacteria in the Southern Ocean with several Flavobacteria clades detected in PFZ and AZ waters not detected in TZ and SAZ waters that putatively represent psychrophilic taxa. Sequence data included a large, so far uncultivated, cosmopolitan phylogenetic clade ("DE cluster 2") that is distributed throughout the Southern Ocean.     

Effects of incubation temperature on growth and production of exopolysaccharides by an antarctic sea ice bacterium grown in batch culture

The sea ice microbial community plays a key role in the productivity of the Southern Ocean. Exopolysaccharide (EPS) is a major component of the exopolymer secreted by many marine bacteria to enhance survival and is abundant in sea ice brine channels, but little is known about its function there. This study investigated the effects of temperature on EPS production in batch culture by CAM025, a marine bacterium isolated from sea ice sampled from the Southern Ocean. Previous studies have shown that CAM025 is a member of the genus Pseudoalteromonas and therefore belongs to a group found to be abundant in sea ice by culture-dependent and -independent techniques. Batch cultures were grown at -2 degrees C, 10 degrees C, and 20 degrees C, and cell number, optical density, pH, glucose concentration, and viscosity were monitored. The yield of EPS at -2 degrees C and 10 degrees C was 30 times higher than at 20 degrees C, which is the optimum growth temperature for many psychrotolerant strains. EPS may have a cryoprotective role in brine channels of sea ice, where extremes of high salinity and low temperature impose pressures on microbial growth and survival. The EPS produced at -2 degrees C and 10 degrees C had a higher uronic acid content than that produced at 20 degrees C. The availability of iron as a trace metal is of critical importance in the Southern Ocean, where it is known to limit primary production. EPS from strain CAM025 is polyanionic and may bind dissolved cations such at trace metals, and therefore the presence of bacterial EPS in the Antarctic marine environment may have important ecological implications.     

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.     

Functional annotation of the putative orphan Caenorhabditis elegans G-protein-coupled receptor C10C6.2 as a FLP15 peptide receptor

This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [35S]guanosine 5'-O-(thiotriphosphate) (GTPgammaS) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B > F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTPgammaS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 degrees C. However, a 37 to 28 degrees C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.     

Nutritional quality of prebreeding diet influences breeding performance of the Florida scrub-jay

Food supplementation studies of breeding birds have traditionally concentrated on energetic constraints on breeding performance. It is only recently that the nutritional quality of the prebreeding diet has also been considered influential. We examined the importance of specific nutrients in the prebreeding diet of the Florida scrub-jay ( Aphelocoma coerulescens). Birds were provided with one of two supplements (rich in protein and fat or rich in fat only) prior to breeding in 2000 and 2001 and their breeding performance, in relation to unsupplemented (control) birds, was examined. Birds receiving both supplements significantly advanced laying in both years, and increased clutch size in 2000 but not in 2001. Laying date explained variation in clutch size in birds on dietary supplements. Egg mass and volume declined with laying order, irrespective of dietary treatment, but birds on the high fat, high protein diet laid heavier third eggs than controls and this was independent of laying date. Laboratory analysis of 14 abandoned and unhatched eggs revealed that as egg mass increased so did the absolute amount of protein and water while fat content remained relatively fixed. Using these relationships between the masses of egg components and fresh egg mass, we calculated that heavier third eggs laid by birds on high fat and high protein, compared with those laid by controls, contained more water that may be fundamental to chick growth and survival. This is the first demonstration for an avian species that nutritional quality of prebreeding diet can simultaneously influence laying date, clutch size, and egg size and composition.     

CCL27-CCR10 interactions regulate T cell-mediated skin inflammation

The skin-associated chemokine CCL27 (also called CTACK, ALP and ESkine) and its receptor CCR10 (GPR-2) mediate chemotactic responses of skin-homing T cells in vitro. Here we report that most skin-infiltrating lymphocytes in patients suffering from psoriasis, atopic or allergic-contact dermatitis express CCR10. Epidermal basal keratinocytes produced CCL27 protein that bound to extracellular matrix, mediated adhesion and was displayed on the surface of dermal endothelial cells. Tumor necrosis factor-alpha and interleukin-1beta induced CCL27 production whereas the glucocorticosteroid clobetasol propionate suppressed it. Circulating skin-homing CLA+ T cells, dermal microvascular endothelial cells and fibroblasts expressed CCR10 on their cell surface. In vivo, intracutaneous CCL27 injection attracted lymphocytes and, conversely, neutralization of CCL27-CCR10 interactions impaired lymphocyte recruitment to the skin leading to the suppression of allergen-induced skin inflammation. Together, these findings indicate that CCL27-CCR10 interactions have a pivotal role in T cell-mediated skin inflammation.     

A mini-column method for routine measurement of human prostatic androgen receptors

The complex heterogeneous nature of the human prostate gland is such that it is advisable to know the histological characteristics of each sample used for androgen receptor (AR) measurement. Adequate size of sample for AR determination is thus a problem if specimens provided during routine transurethral prostatectomy are to be used for both estimation of AR and histological examination. We present a simple method suitable for these small specimens in which [3H]R 1881 bound to AR is separated from free steroid on mini-columns of controlled-pore glass beads. Data obtained indicate a single class of binding sites of high affinity and low capacity with steroid specificity typical of an androgen receptor. The assay is suitable for samples as small as 20 mg wet weight and is linear using 25-125 microliter cytosol (correlation coefficient 0.995). Intra-assay variation is 6.8% and interassay variation 25.8% (n = 22) over 4 months. A single saturating concentration of steroid measures 97% of AR calculated by Scatchard analysis. Inclusion of high salt (0.4 M KNO3) and 10 mM dithiothreitol in incubation buffer at pH 8.4 are essential; inclusion of 10 mM sodium molybdate in the homogenisation buffer improves measurement. A comparison of AR measured in histologically similar samples obtained by a transurethral resectoscope (TUR) and a cold punch resectoscope (CPR) taken in juxtaposition demonstrated no difference in receptor content. Although carcinomatous samples contained significantly higher receptors levels than benign samples, no differences were observed between TUR and CPR specimens.     

Seasonal changes in tissue elasticity in chaparral shrubs

An important physiological feature of chaparral shrubs is the development of low water potentials during periods of drought characteristic of southern Californian summers. Changes in tissue elasticity may be an important characteristic allowing these low water potentials to be reached and maintained without the development of detrimental water deficits. To examine this possibility, seasonal changes in tissue elasticity were measured in 3 species of chaparral shrubs, Arctostaphylos glandulosa Eastw., Quercus dumosa Nutt. and Ceanothus greggii Gray., by the pressure-volume method. Tissue elasticity was characterized using graphs of the modulus of elasticity plotted as a function of turgor pressure, and maximum values of the elastic modulus. The moduli of elasticity of the shrubs increased following leaf emergence in the spring, were highest during periods of low soil water potential, and tended to decrease following the summer-fall drought period. Increases in tissue elasticity facilitate water uptake from drying soils, but result in greater turgor loss during tissue dehydration.