Characterization of 25-hydroxyvitamin D binding protein from intestinal cells

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.     

Copepod feeding response to varying Alexandrium spp. cellular toxicity and cell concentration among natural plankton samples

We challenged four species of copepod grazers (Acartia hudsonica, Centropages hamatus, Eurytemora herdmani, Calanus finmarchicus) with natural water samples containing non-toxic algae mixed with one of three clones of Alexandrium spp.—A. tamarense GTCN16 (non-toxic), A. fundyense GTCA28 (moderate toxicity), and A. fundyense BC1 (higher toxicity), each at relatively high (10⁵ cells L⁻¹) and low (10⁴ cells L⁻¹) concentrations. Within any one copepod species, significant differences existed in copepod clearance rates and total food ingested between high and low Alexandrium cell concentrations, and between levels of toxicity, but feeding response did not follow a predictable relationship proportional to toxin levels—rather, the presence or absence of toxin was more important than the level of toxicity. C. finmarchicus behaved differently from the smaller coastal copepods, showing less selectivity and greater concentration dependence. In low Alexandrium concentration treatments, copepod clearance rates on Alexandrium were usually higher, and electivity indices for Alexandrium less negative (indicating less avoidance), compared to high concentration treatments. In high toxicity (BC1) treatments of all copepod species (except C. finmarchicus), total food consumption was consistently less at high Alexandrium concentrations compared to low concentrations, suggesting that high toxicity and concentration suppress overall feeding, while in non-toxic (GTCN 16) treatments total consumption was always higher at high Alexandrium concentrations. Copepod grazers do not follow predictable feeding rules throughout a continuum of conditions, but become more predictable at extremes of concentration and toxicity of prey, consistent with the conclusion that both factors are important. Results support the hypothesis that grazer deterrence imparted by toxicity is only effective at high cell concentrations, but even then will not protect against all grazers.     

Selecting representative model micro-organisms

Background

The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.

Results

The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.

Conclusion

This archived set of Gateway^® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.     

Adrenaline Increases Cyclic 3′5′-AMP Formation in Hamster Epidermis

CATECHOLAMINES probably influence cell proliferation by delaying cells in the premitotic phase^1,2. Bullough and Laurence found that crude skin extracts contained a tissue-specific protein (chalone) which inhibited epidermal cell proliferation and that the action of this extract was augmented by adrenaline³. They later found that adrenaline alone (0.00025 µg/ml.) reduced epidermal mitotic activity in mouse ears by about 50% in vitro⁴.     

Some virus and virus-like diseases of tobacco,tomato, papaya,and rubber tree in vietnam and cambodia

In Vietnam a green strain of tobacco mosaic virus was isolated having TIP 89°C (10 min) and causing systemic necrosis in tobacoo ‘Xanthi-nc’ and sometimes also inDatura stramonium. In symptomless tomato plants an elongated virus belonging apparently to the Carlavirus group (NL 630 nm) was found. In papaya trees showing severe symptoms of mosaic and/or ringspot elongated virus particles (NL 730 nm) were observed; this virus being apparently a member of the Potyvirus group, resembled as far as its symptoms in papaya are concerned, the papaya ringspot or the distortion ringspot. In Cambodia some young rubber trees showed malformed leaves (esp. edges and veins) with yellow discolorations along the veins. Such leaves contained elongated virus-like particles (rigid or slightly flexible) of various length (60 to 880 nm), so that their normal length (NL) could not be established precisely. Particles 120 to 150 nm long occurred very frequently.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Allometry between leg and body length of insects: lack of support for the size–grain hypothesis

• 1 The size–grain hypothesis ( Kaspari & Weiser, 1999 ) states that (1) as organisms decrease in size, they perceive their environment as being more rugose; (2) long legs allow organisms to step over obstacles but hinder them from entering small gaps; and (3) as the size of an organism decreases, the benefits of long legs begin to be outweighed by the costs of construction. Natural selection should therefore favour proportionally longer legs in larger organisms, thereby leading to a positive allometry between leg and body length (scaling exponent b > 1).
• 2 Here we compare the scaling exponent of leg‐to‐body length relationships among insects that walk, walk and fly, and predominantly fly. We measured the lengths of the hind tibia, hind femur, and body length of each species.
• 3 The taxa varied considerably in the scaling exponent b. In seven out of ten groups (Formicidae, Isoptera, Carabidae, Pentatomidae, Apidae, Lepidoptera, Odonata adult), b was significantly greater than one. However, there was no gradual decrease in b from walking to walking/flying to flying insects.
• 4 The results of the present study provide no support for the size–grain hypothesis. We propose that leg length is not only affected by the rugosity of the environment, but also by (1) functional adaptations, (2) phylogeny, (3) lifestyle, (4) the type of insect development (hemimetabolism or holometabolism), and (5) constraints of gas exchange.

     

PARAMETERS USED FOR RECOGNITION OF DISTRESS CALLS IN TWO SPECIES: LARUS ARGENTATUS AND STURNUS VULGARIS

ABSTRACT

Distress calls are signals effective over a long distance. They are well known to evoke interspecific reactions. We suggest that the interspecificity phenomenon results from the use of similar laws of decoding by the species concerned. These laws must take into account the transmission channel which always has a great influence on long-range communication. We tested our hypothesis by broadcasting simplified synthetic calls to two species of birds: the herring gull and the starling. The various calls differed in terms of frequency modulation (FM). Two main conclusions emerged from this series of tests:

1. The parameters used for recognition are not sophisticated: a simple slope applied to a carrier frequency that corresponds to the acoustic shape of a distress call is sufficient to confer a distress meaning to the signal. The basic rules are the same for the gull and the starling, with differences only in the acceptance level of the species.

2. The system of recognition is based upon parameters not altered by the environment: the birds make use of the slow frequency modulations (FMI). In contrast, the fast frequency sweeps (FMII) which are modified during propagation do not seem to be utilised. The use of these characteristics of distress calls for recognition allows interspecificity and maximum efficiency for propagation over long distances.