Growth and tissue mass cycles in the infaunal bivalve Yoldia eightsi at Signy Island, Antarctica

Shell growth in Yoldia eightsi was measured over an austral summer and winter in 1992. In specimens < 12 mm length, growth was not significantly different between summer and winter periods, and the fastest recorded rate, 6.3 μm day⁻¹ was for 5-mm individuals during the winter. In summer, specimens of all lengths grew significantly, but in winter bivalves > 27 mm length did not increase in length. Tissue dry and ash-free dry mass (AFDM) cycles were assessed at monthly intervals between December 1988 and January 1991. ANCOVA indicated significant interannual and seasonal effects on this cycle. Tissue mass increased in the summer, coinciding with the phytoplankton bloom and the period of maximum sedimentation of organic material from the water column. A standard 20-mm-length animal reached a maximum AFDM of 114 mg in February 1990. The minimum value (68 mg AFDM) throughout the 2 years of measurements was in early December 1988, at the end of the austral winter. Periods of tissue mass increase were, therefore, decoupled from shell growth, at least in juveniles. Tissue mass was significantly higher in 1990 than 1989, which was mainly due to high organic contents in the summer (January to May). This was not consistent with the pattern of organic content in the sediments at the study site, but was in phase with the cycle in sediment chlorophyll a content. Tissue mass increase depended on major resource input during the summer, but Y. eightsi was capable of maintaining winter condition from stocks of benthic microalgae in years of poor ice cover. Tissue mass declined between April and July each year. This was accompanied by large falls in tissue ash content, and coincided with the spawning period in early June. These are the first monthly tissue mass data collected over a 2-year period for an Antarctic mollusc. They are the first such data indicating seasonal variation in tissue mass and showing a decoupling of shell and tissue growth in a polar bivalve. The P/B ratio calculated from these data was 0.106, which is slightly lower than previous values found for this species, but is in line with general values for Antarctic marine benthos. Accepted: 6 December 1999     

Purification of Primordial Germ Cells from TNAPMouse Embryos Using FACS-gal

Tissue nonspecific alkaline phosphatase (TNAP), the product of theAkp2locus, is expressed in mouse primordial germ cells (PGC) for an extensive period during embryogenesis. Mice with theAkp2^tm1Sormutant allele of TNAP expresslacZ(β-galactosidase; β-gal) under control of theAkp2locus. PGCs were purified fromAkp2^tm1Sorembryos using fluorescence activated cell sorting of β-gal expressing cells (FACS-gal). Analysis of the purified cells by alkaline phosphatase staining and immunocytochemistry with anti-c-kitantibody demonstrated that highly (98%) purified PGCs can be isolated using this method. This technique will facilitate experiments that require highly purified preparations of PGCs including cell culture and gene expression analyses.     

Lead Optimization of N 6-Cyclopentyl-3′-amido-3′-deoxyxylofuranosyladenines as Adenosine A1 Receptor Antagonists

Abstract

Strategies toward the further lead optimization of N ⁶-cyclopentyl-3′-amido-3′-deoxyxylofuranosyladenosines as adenosine A₁ receptor antagonists including the synthesis of the 5′-deoxy-analogues and a practical method for parallel amidation are presented.     

Isolation and characterization of an adriamycin-resistant breast tumor cell line

Summary An adriamycin-resistant human breast tumor cell line MDA-A1 ^R was generated by step-wise selection in increasing concentrations of drug from the parent cell line MDA-MB-231. MDA-A1 ^R cells grow as loosely attached cell aggregates with a doubling time of 28–32 h; the MDA-MB-231 parent cell line grows as a standard monolayer culture with a 20-h doubling time. The MDA-A1 ^R cell line is highly resistant to adriamycin compared to the parent cell line, and is cross-resistant to velban and colchicine suggestive of a multidrug resistance (MDR) phenotype. MDA-A1 ^R cells exhibit reduced net adriamycin conent as compared to the parent cell line. The MDR-associated P-glycoprotein gene is amplified approximately 10-to 30-fold in MDA-A1 ^R cells. P-glycoprotein sequences are overexpressed in the resistant cells and are stable for up to 13 wk after drug removal. Moreover, MDA-A1 ^R cells show the presence of very high levels of P-glycoprotein. MDA-A1 ^R is thus an in vitro model system to study the mechanism of MDR in human breast cancer. This work was supported in part by grant C30195 from the National Institute of health, Bethesda, MD. Portion of this study appeared as a poster presentation at the Tissue Culture Association meeting, Las Vegas, 1988.     

Terrestrial vegetation at Canada Glacier,Southern Victoria Land,Antarctica

Summary Bryophyte flushes in the vicinity of Canada Glacier in S.S.S.I. No. 12, Taylor Valley, Southern Victoria Land, were investigated in order to describe the vegetation present and to investigate factors affecting vegetation distribution. Biomass values from 950 to 1,250 g m^–2 (dry weight) and vegetated areas up to 14,450 m² indicate this is a significant area of bryophyte growth in Southern Victoria Land. The pattern of plant species in relation to water flow was investigated through detailed mapping. This is the first confirmed identification of Bryum argenteum, and Pottia heimii, and the first report of Bryum pseudotriquetrum from this area. Tissue nitrogen values for plants at this site are lower than other reported data, but it seems unlikely this would be a limiting factor for growth. It is concluded that, in this area, summer water flow in conjunction with microtopography has the greatest influence in determining where mosses, cyanobacteria and algae grow.     

Scleroglucan and oxalic acid formation by Sclerotium glucanicum in sucrose supplemented fermentations

Summary Strategies for adding sucrose to stationary phase cultures of Sclerotium glucanicum to improve scleroglucan production were examined. Particular attention was paid to the effect of sucrose supplementation on the formation of the toxic by-product, oxalic acid. The rate of addition of sucrose was found to markedly influence the outcome of the process, with addition at a rate of 0.084 gl^–1h^–1 giving a 50% improvement in broth biopolymer concentration. Additions at higher rates seemed to cause inhibition of the culture. However, greatly increased oxalic acid concentrations in the sucrose supplemented fermentations, and the impact of unfavourable changes in productivity and specific productivity call into question the utility of this proposed method of process improvement for this microorganism.     

Eradication of rabbits from landscape scale exclosures: pipedream or possibility?

Summary High densities of the European Rabbit (Oryctolagus cuniculus) were eradicated from 60 km² of the Arid Recovery Reserve between 1996 and 2001. Eradication was possible due to an initial knockdown caused by rabbit haemorrhagic disease, followed by effective exclusion fencing, broadscale poison baiting, targeted shooting, warren destruction and trapping on rabbit burrows and buckheaps. The efficacy of different broadscale control and intensive rabbit eradication techniques was subsequently estimated in a 26 km² expansion to the Reserve from 2002 to 2006. Non‐target implications of these control techniques were also assessed where possible. An estimated 8000 rabbits were removed in total from both areas and results suggest that rabbit eradication is possible at a landscape scale. Strategies for eliminating rabbits from confined areas are suggested.     

Murine antigen H-2.7: Its genetics,tissue expression,and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2 ^an1 ), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2 ^an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.Abbreviations used in this paper ACT Ammonium chloride Tris-buffer - BSA Bovine serum albumin - HA Hemagglutination - HASI Hemagglutination serum inhibition - HBSS Hanks balanced salt solution - PBS Phosphate buffered saline - PVP Polyvinylpyrrolidone - RBCs Red blood cells     

Method for enzymatic determination of imidazole acetic acid

A method for enzymatic assay of imidazole acetic acid (ImAA) was developed, based on the strict substrate specificity of imidazole acetate monooxygenase from Pseudomonas sp. [Maki et al. (1969) J. Biol. Chem., 244., 2942–2950], which catalyzes concomitant conversion of NADH to NAD⁺. Thus, ImAA was determined by measuring decrease in absorbancy at 340 nm. Tissue extracts were partially purified and/or concentrated by column chromatography on Bio-Rad AG-1 before enzymatic assay. The lowest measurable level of ImAA by this method was 2 nmol.     

CCN2 (Connective Tissue Growth Factor) is essential for extracellular matrix production and integrin signaling in chondrocytes

The matricellular protein CCN2 (Connective Tissue Growth Factor; CTGF) is an essential mediator of ECM composition, as revealed through analysis of Ccn2 deficient mice. These die at birth due to complications arising from impaired endochondral ossification. However, the mechanism(s) by which CCN2 mediates its effects in cartilage are unclear. We investigated these mechanisms using Ccn2 ^−/− chondrocytes. Expression of type II collagen and aggrecan were decreased in Ccn2 ^−/− chondrocytes, confirming a defect in ECM production. Ccn2 ^−/− chondrocytes also exhibited impaired DNA synthesis and reduced adhesion to fibronectin. This latter defect is associated with decreased expression of α5 integrin. Moreover, CCN2 can bind to integrin α5β1 in chondrocytes and can stimulate increased expression of integrin α5. Consistent with an essential role for CCN2 as a ligand for integrins, immunofluorescence and Western blot analysis revealed that levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 phosphorylation were reduced in Ccn2 ^−/− chondrocytes. These findings argue that CCN2 exerts major effects in chondrocytes through its ability to (1) regulate ECM production and integrin α5 expression, (2) engage integrins and (3) activate integrin-mediated signaling pathways.     

Effects of vesicular-arbuscular mycorrhizae on growth and phosphorus and zinc nutrition of peanut (Arachis hypogaea L.) in an Oxisol from subtropical Australia

Peanut plants (cv. Shulamit) were grown in an Oxisol soil in pots in the glasshouse to assess effects of soil sterilization and inoculation with spores of vesicular-arbuscular mycorrhizal fungi (VAMF) on the response to five rates of phosphorus (0 to 240 kg P ha^–1) and two rates of zinc (0 and 10 kg Zn ha^–1) fertilizers.Both P and Zn nutrition were affected by VAMF activity but the dominant role of VAMF in this soil type was in uptake of P. In the absence of VAMF there was a clear threshold level of P application (60 kg P ha^–1) below which plants grew poorly, which resulted in a sigmoidal response of dry matter to applied P. The maximum response was not fully defined because dry matter production continued to increase up to 240 kg P ha^–1. Tissue P concentration of non-mycorrhizal plants increased linearly with P rate and was always significantly less than that in mycorrhizal plants.Mycorrhizal plants responded without threshold to increasing P rate, attaining maximum dry matter at 120 kg P ha^–1 in inoculated sterilized soil and at 30 kg P ha^–1 in nonsterile soil. These differences in maximal P rates and in the greater dry matter produced in sterile soil at high P rates were attributed to the negative effects of the root-knot nematodeMeloidogyne hapla in nonsterile soil.Plant weight did not respond to zinc fertilizer but tissue Zn concentration increased with applied Zn. Tissue Zn concentration and uptake were increased by VAMF.     

Murine “housekeeping” enzyme (genetic locus:Idh-1) is regulated in an allele-specific manner

The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1^a allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1^a allele. While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.     

Lethal effects of Gliocladium virens,Beauveria bassiana and Metarhizium anisopliae on the malaria vector Anopheles albimanus (Diptera: Culicidae)

Control of Anopheles albimanus, the main vector of malaria on the coast of the State of Chiapas, is based mainly on application of chemical insecticides, which has resulted in resistance to most registered insecticides. Strategies for biological control may provide sustainable alternatives. We report on the lethal effects of a native isolate of Gliocladium virens on An. albimanus larvae and adults, compared to that of strains of Beauveria bassiana and Metarhizium anisopliae. Conidial suspensions of G. virens, B. bassiana and M. anisopliae cultured on Sabouraud agar were tested in bioassays with An. albimanus larvae and adults. Mosquito larvae were more susceptible to all fungi, compared to adults. On early and late instar larvae, M. anisopliae showed the most pathogenic effect (LC₅₀ of 1.4×10⁵ conidia/mL in early instars; 1.1×10⁵ conidia/mL in late instars), followed by G. virens (LC₅₀ of 3.3×10⁵ conidia/mL in early instars and 3.5×10⁶ conidia/mL in late instars). Metarhizium anisopliae sensu lato and the native G. virens could be considered good choices for An. albimanus control in southern Mexico.     

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H⁺-ATPases. We investigated expression of Lycopersicon esculentum Mill. H⁺-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H⁺-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.     

Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine

In tubular epithelia, barrier function varies in a segment-specific way. The aim of this study was to correlate the presence of tight junction proteins and paracellular barrier properties along rat intestine. Tissue segments of duodenum, jejunum, ileum, and colon were stripped of submucosal cell layers and mounted in Ussing chambers for impedance spectroscopy to measure epithelial resistance (R ^epi). In parallel, expression of tight junction proteins was analysed by Western blots and immune fluorescence confocal microscopy. Colon showed highest R ^epi, followed by duodenum, jejunum, and ileum. In small intestine, common transepithelial resistance (R ^trans or TER) overestimated true R ^epi by ~60%. In colon, strongest expression of “tightening” claudins 1, 3, 4, 5, and 8 was detected. In accordance with R ^epi the most proximal of the small intestinal segments, duodenum exhibited highest expression of “tightening” claudins and lowest expression of claudins mediating permeability, namely claudin-2, -7, and -12, compared to jejunum and ileum. These results correspond to the specific role of the duodenum as the first segment facing the acidic gastric content.     

Chemiluminometric β-galactosidase detection as a basis for a tetracycline screening test in milk

The observation that tetracyclines inhibit the biosynthesis of β-galactosidase in Escherichia coli to a greater extent than other antibacterials was exploited for the development of a chemiluminometric method to detect residues of this class of antibiotics in milk. The procedure involves the incubation of a milk sample with 10⁷ CFU/ml of an E. coli strain in the presence of IPTG, an inducer of β-galactosidase, and of EGTA, a chelator of calcium ions, followed by a 1000-fold dilution and measurement of the residual enzymatic activity using the chemiluminogenic substrate Galacton. Chemiluminometry proved an essential tool in this procedure because the extensive dilution of the sample, necessary to avoid light quenching by turbidity, results in an insufficient level of β-galactosidase activity to be measurable by colorimetry. This tetracycline galactosidase (TG) test has been validated and compared in the field to existing commercial screening assays for antibiotics. Its detection limit for tetracyclines ranges between 40 and 65 μg/kg, which is below the European maximum residue limit (MRL = 100 μg/kg) in milk. No other antibacterials, at concentrations commonly expected in milk, were found to interfere with the TG test. Strategies to avoid false positive reactions possibly arising from very high somatic cell counts will be reported elsewhere. © 1998 John Wiley & Sons, Ltd.     

Productivity and selective accumulation of carotenoids of the novel extremophile microalga <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> grown with different carbon sources in batch systems

Cultivation of extremophile microorganisms has attracted interest due to their ability to accumulate high-value compounds. Chlamydomonas acidophila is an acidophile green microalga isolated by our group from Tinto River, an acidic river that flows down from the mining area in Huelva, Spain. This microalga accumulates high concentrations of lutein, a very well-known natural antioxidant. The aim of this study is to assess use of different carbon sources (CO₂, glucose, glycerol, starch, urea, and glycine) for efficient growth of and carotenoid production by C. acidophila. Our results reveal that growth of the microalga on different carbon sources resulted in different algal biomass productivities, urea being as efficient as CO₂ when used as sole carbon source (~20 g dry biomass m^–2 day^–1). Mixotrophic growth on glucose was also efficient in terms of biomass production (~14 g dry biomass m^–2 day^–1). In terms of carotenoid accumulation, mixotrophic growth on urea resulted in even higher productivity of carotenoids (mainly lutein, probably via α-carotene) than obtained with photoautotrophic cultures (70% versus 65% relative abundance of lutein, respectively). The accumulated lutein concentrations of C. acidophila reported in this work (about 10 g/kg dry weight, produced in batch systems) are among the highest reported for a microalga. Glycerol and glycine seem to enhance β-carotene biosynthesis, and when glycine is used as carbon source, zeaxanthin becomes the most accumulated carotenoid in the microalga. Strategies for production of lutein and zeaxanthin are suggested based on the obtained results.