Nitrogen mineralisation and plant nitrogen acquisition in a nitrogen-limited calcareous grassland

A field study measured the rate of soil mineral N supply and its effects on plant biomass and N accumulation in a 13-year-old, naturally regenerating, calcareous grassland. Gross rates of N mineralisation (2 μg g⁻¹ day⁻¹, i.e. 0.69 kg ha⁻¹ day⁻¹), assessed using ¹⁵N pool dilution, were at the lower end of the range previously reported for grasslands. Weekly additions of liquid N fertiliser ([NH₄]₂SO₄, NH₄NO₃ or KNO₃) and, to a lesser extent the addition of water, increased plant growth substantially, demonstrating that the primary constraint to plant growth was low N availability. In plants that had received NO₃⁻, the activity of the inducible enzyme nitrate reductase in shoots initially increased in proportion to the amount of NO₃⁻ supplied. However, as above-ground herbage accumulated, nitrate reductase activity declined to similar low levels in all treatments, despite the continuance of the constant NO₃⁻ additions. The decline in NR specific activity reflected declining tissue NO₃⁻ concentrations, although total plant NRA may have remained constant during the period of study. The study has shown that plant growth is limited by low N mineralisation rates and indeed the soil is a sink for much added N. Low water availability provides an additional constraint on N mineralisation in this calcareous grassland soil. Any disturbances in the N cycle which increase the availability of mineral N will result in a substantial increase in plant growth within this ecosystem.     

Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells

A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD) that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag) and BCGΔpanCD[pHS400] (BCGpan-Gag) were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag). After the boost high frequencies of predominantly Gag-specific CD8(+) T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4(+) T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.     

Rigidification of the autolysis loop enhances Na(+) binding to thrombin

Binding of Na⁺ to thrombin ensures high activity toward physiological substrates and optimizes the procoagulant and prothrombotic roles of the enzyme in vivo. Under physiological conditions of pH and temperature, the binding affinity of Na⁺ is weak due to large heat capacity and enthalpy changes associated with binding, and the K_d = 80 mM ensures only 64% saturation of the site at the concentration of Na⁺ in the blood (140 mM). Residues controlling Na⁺ binding and activation have been identified. Yet, attempts to improve the interaction of Na⁺ with thrombin and possibly increase catalytic activity under physiological conditions have so far been unsuccessful. Here we report how replacement of the flexible autolysis loop of human thrombin with the homologous rigid domain of the murine enzyme results in a drastic (up to 10-fold) increase in Na⁺ affinity and a significant improvement in the catalytic activity of the enzyme. Rigidification of the autolysis loop abolishes the heat capacity change associated with Na⁺ binding observed in the wild-type and also increases the stability of thrombin. These findings have general relevance to protein engineering studies of clotting proteases and trypsin-like enzymes.     

Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.     

Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UAS_E) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.     

Hunting strategies in wild common marmosets are prey and age dependent

We investigated the hunting strategies of wild common marmosets (Callithrix jacchus) to determine whether the strategies differed among animals of different age classes and/or prey type. The study was conducted in a fragment of Atlantic Rain Forest, situated 40 km from Recife (PE/Brazil). Twenty‐seven individuals from four social groups were observed. Captured prey items were divided into three categories. The hunting strategies of the common marmosets were ranked into four categories. The acquisition of larger prey (items more than 2.0 cm) involved the appropriate body movements and postures that concealed the approaching marmosets, whereas the acquisition of smaller prey (items under 2.0 cm) involved less concealing behaviors. Furthermore, adults and juveniles (age ≥5 months) were more capable of capturing larger prey than were younger (1–2 months) or older infants (3–4 months). Although older infants were successful in capturing certain prey, they often failed when they attempted to capture larger prey that jumped and/or used flight to escape. The results suggest that both the experience of the monkeys and escape behavior of the prey affect predation efficiency in wild common marmosets. Am. J. Primatol. 72:1039–1046, 2010. © 2010 Wiley‐Liss, Inc.     

Levonorgestrel antagonism on estrogen-induced pituitary tumors is mediated by progesterone receptors

Using both IN VITRO and IN VIVO approaches, we studied the antagonism exerted by the synthetic progestin levonorgestrel on estrogen-induced prolactinomas, considering that levonorgestrel shows partial androgenic properties and that androgens inhibit estrogen-induced prolactin synthesis and secretion. In the tumors, binding of estrogens to their receptors was competed neither by progesterone receptor ligands nor by androgen receptor ligands, ruling out direct inhibitory effects of these drugs on tumor development. Progestin binding was competed by the progesterone receptor agonists progesterone and levonorgestrel, by the antagonist mifepristone, and also by the androgen dihydrotestosterone, whereas the androgen receptor antagonist hydroxyflutamide was a weak competitor. In addition, both progesterone receptor and androgen receptor ligands competed for binding to androgen receptors. In primary cultures of pituitary tumors, levonorgestrel decreased prolactin secretion, an effect that was blocked by mifepristone but not by hydroxyflutamide. IN VIVO results indicated that levonorgestrel inhibition of both estrogen-induced pituitary weight increment and hyperprolactinemia was reduced by mifepristone, whereas flutamide was unable to block levonorgestrel effects. Our results suggest that even when an interaction of levonorgestrel with androgen receptors in the tumors is possible, the antagonistic effects of levonorgestrel on tumor development and functionality are mediated by progesterone receptors.     

Perspective: the evolution of warning coloration is not paradoxical

Animals that are brightly colored have intrigued scientists since the time of Darwin, because it seems surprising that prey should have evolved to be clearly visible to predators. Often this self-advertisement is explained by the prey being unprofitable in some way, with the conspicuous warning coloration helping to protect the prey because it signals to potential predators that the prey is unprofitable. However, such signals only work in this way once predators have learned to associate the conspicuous color with the unprofitability of the prey. The evolution of warning coloration is still widely considered to be a paradox, because it has traditionally been assumed that the very first brightly colored individuals would be at an immediate selective disadvantage because of their greater conspicuousness to predators that are naive to the meaning of the signal. As a result, it has been difficult to understand how a novel conspicuous color morph could ever avoid extinction for long enough for predators to become educated about the signal. Thus, the traditional view that the evolution of warning coloration is difficult to explain rests entirely on assumptions about the foraging behavior of predators. However, we review recent evidence from a range of studies of predator foraging decisions, which refute these established assumptions. These studies show that: (1) Many predators are so conservative in their food preferences that even very conspicuous novel prey morphs are not necessarily at a selective disadvantage. (2) The survival and spread of novel color morphs can be simulated in field and aviary experiments using real predators (birds) foraging on successive generations of artificial prey populations. This work demonstrates that the foraging preferences of predators can regularly (though not always) result in the increase to fixation of a novel morph appearing in a population of familiar-colored prey. Such fixation events occur even if both novel and familiar prey are fully palatable and despite the novel food being much more conspicuous than the familiar prey. These studies therefore provide strong empirical evidence that conspicuous coloration can evolve readily, and repeatedly, as a result of the conservative foraging decisions of predators.     

A novel artemisinin-quinine hybrid with potent antimalarial activity

Artemisinin was reduced to dihydroartemisinin and coupled to a carboxylic acid derivative of quinine via an ester linkage. This novel hybrid molecule had potent activity against the 3D7 and (drug-resistant) FcB1 strains of Plasmodium falciparum in culture. The activity was superior to that of artemisinin alone, quinine alone, or a 1:1 mixture of artemisinin and quinine.