Purification and characterization of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus.

We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-1 was expressed in Sf9 insect cells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding. ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.     

C-Terminally PEGylated hGH-derivatives

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.     

Metallothionein levels in willow ptarmigan (<Emphasis Type="Italic">Lagopus lagopus</Emphasis>) populations with different natural loads of cadmium

The tissues of willow ptarmigan in some Norwegian mountain areas contain elevated concentrations of cadmium (Cd). It is not known whether such high Cd levels would have negative impacts in otherwise healthy populations of this species. The aim of the current study was to clarify relationships between hepatic and renal metallothionein (MT) and Cd concentrations in willow ptarmigan to assess effects from this metal. The study reported here was undertaken on willow ptarmigan from the Kongsvoll area, with a naturally high Cd load, and the Essand area, with a naturally low Cd load. Cd values in liver and kidney in willow ptarmigan from Kongsvoll were significantly higher than in willow ptarmigan from Essand. The MT content in both tissues was also highest in willow ptarmigan from Kongsvoll. The MT concentration in kidney was twice that in liver for ptarmigan from both areas and at all times of the year. The MT level in both liver and kidney varied greatly throughout the season, with the highest content in spring (May). The variation was greatest in liver. The total material showed a significant linear relationship between Cd and MT levels in both liver and kidney, but a breakdown of the material into seasons and areas gave a varying degree of significance. MT in willow ptarmigan may be an important mechanism for detoxifying Cd, and populations exposed to high load may “respond” to the loads by increasing MT synthesis in tissues such as liver and kidney. There is no evidence that willow ptarmigan from areas with high natural Cd loads have reached a limit for MT synthesis in either liver or kidney.     

Sensitivity of early mouse embryos to [H]thymidine

Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to [³H]thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of [³H]thymidine, concentrations as low as 0.1 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs was reduced, and at 10 nCi/ml the number of embryos developing to all three post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highest concentration tested (100 nCi/ml). When embryos were cultured from the 2-cell stage for 3 days in the presence of [³H]thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to [³H]thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages; the lowest concentration that interfered with development was 10 nCi/ml, and exposure at the morula stage was most detrimental to the subsequent development of embryos, particularly that of ICMs. The 24-h exposure of immunosurgically isolated ICMs to [³H]thymidine revealed that the high sensitivity of the ICM to [³H]thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate [³H]thymidine into the DNA.     

Salinity Induced Regime Shift in Shallow Brackish Lagoons

In brackish lagoons, Daphnia is replaced by calanoid copepods (Eurytemora affinis, Acartia spp.) and rotifers when a certain threshold (depending on, for instance, fish density) is reached. We hypothesize that loss of Daphnia induces a regime shift from clear to turbid at high nutrient concentrations. We conducted a factorial designed enclosure experiment with contrasting salinities (0–16‰), low fish predation (one three-spined stickleback, Gasterosteus aculeatus, m⁻²) and three levels of nutrient loading in a shallow brackish lagoon. A change point analysis suggests a strong regime shift from a clear to a turbid state at 6–8‰ salinity at low and high loading, but not for the control. From the low to the high salt regime, chlorophyll a (Chla), Chla:total phosphorus (TP) and Chla:total nitrogen (TN) ratios shifted highly significantly for all nutrient treatments, and the bacterioplankton production followed the changes in Chla. These changes occurred parallel with a shift from cladoceran and cyclopoid copepod to rotifer dominance. Monitoring data from 60 Danish brackish lagoons show increasing Chla with increasing TP and TN as well as interactive effects of TN and salinity, peaking at intermediate salinity. A relatively weak effect of salinity at low nutrient concentrations and the stronger effect at intermediate high salinity are in accordance with the experimental results. However, these data suggest a lower salinity threshold than in the experiment, which may be explained by a higher fish density. Our results have implications for the management of coastal lagoons both at present and in a future (predicted) warmer climate: (1) improved water quality can be obtained by reducing the nutrient loading or enhancing the freshwater input to a level triggering a shift to Daphnia dominance (typically <2‰), (2) fish manipulation is probably not a useful tool for brackish lagoons, unless the salinity is below the threshold for a potential shift to a clear Daphnia dominated state, and (3) more abrupt changes will expectedly occur in low-saline coastal lagoons at increasing salinity during summer in a future warmer climate.     

Genetic background impacts soluble and cell wall-bound aromatics in <Emphasis Type="Italic">brown midrib</Emphasis> mutants of sorghum

Sorghum (Sorghum bicolor (L.). Moench) BMR-6 and BMR-12 encode cinnamylalcohol dehydrogenase and caffeic acid-O-methyltransferase, respectively. We have evaluated the impact of two bmr alleles, bmr-6 and bmr-12, respectively, on soluble and wall-bound aromatics in near isogenic, wild-type (WT), bmr-6, bmr-12 and double-mutant (DM; bmr-6 and bmr-12) plants in two genetic backgrounds, RTx430 and Wheatland. Immunoblots confirmed that COMT protein was essentially absent in bmr-12 and DM plants, but was present in bmr-6 and WT plants. In contrast, although CAD activity was not detected in bmr-6 and DM plants, proteins crossreacting to anti-CAD sera were found in stem extracts from all genotypes. In both sorghum backgrounds, WT plants had lowest amounts of free aromatics, higher levels of cell wall-bound pCA and FA esters and guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) lignins. Soluble aromatics and cell wall phenolic ester content in Wheatland DM plants resembled that of Wheatland bmr-6 plants, whereas in the RTx430 background, levels of these components in the DM plants more closely resembled those observed in bmr-12 plants. In both backgrounds, bmr-6 plants exhibited reduced levels of G, S, and H lignins relative to WT, and increased incorporation of G-indene into lignin. In bmr-12 plants, there was greater incorporation of G- and 5-hydroxyguaiacyl (5-OHG) lignin into cell walls. Histochemical staining of internode sections from Wheatland plants indicated that apparent lignification of cortical sclerenchyma and vascular bundle fibers was greatest and most uniform in WT plants. Relative staining intensity of these tissues was decreased in bmr-6, followed by bmr-12 plants. DM plants exhibited poor staining of cortical sclerenchyma and vascular bundle fibers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Calmodulin interactions with IQ peptides from voltage-dependent calcium channels

Calmodulin (CaM) functions as a Ca²⁺ sensor for inactivation and, in some cases, facilitation of a variety of voltage-dependent Ca²⁺ channels. A crucial determinant for CaM binding to these channels is the IQ motif in the COOH-terminal tail of the channel-forming subunit. The binding of CaM to IQ peptides from Lc-, P/Q-, and R-type, but not N-type, voltage-dependent Ca²⁺ channels increases the Ca²⁺ affinity of both lobes of CaM, producing similar N- and C-lobe Ca²⁺ affinities. Ca²⁺ associates with and dissociates from the N-lobe much more rapidly than the C-lobe when CaM is bound to the IQ peptides. Compared with the other IQ peptides, CaM-bound Lc-IQ has the highest Ca²⁺ affinity and the most rapid rates of Ca²⁺ association at both lobes, which is likely to make Ca²⁺ binding to CaM, bound to this channel, less sensitive than other channels to intracellular Ca²⁺ buffers. These kinetic differences in Ca²⁺ binding to the lobes of CaM when bound to the different IQ motifs may explain both the ability of CaM to perform multiple functions in these channels and the differences in CaM regulation of the different voltage-dependent Ca²⁺ channels. Ca²⁺-dependent inactivation; Ca²⁺-dependent facilitation; apocalmodulin     

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.     

Hyperlipidaemia and aortic valve disease

PURPOSE OF REVIEW: Degenerative aortic valve stenosis is a common disease in the elderly, and traditional risk factors for atherosclerotic disease including hyperlipidaemia have been associated with the condition in several studies. This review addresses the role of the various risk factors and the potential for intervention. RECENT FINDINGS: The association of lipid abnormalities such as high lipoprotein(a) levels and the presence of the apolipoprotein E4 allele with aortic stenosis, as well as the presence of several inflammatory markers both in plasma and in surgically excised valves, suggest that the stenotic process is driven by many of the same factors behind atherosclerosis. The aortic valves of animals fed a cholesterol-rich diet exhibit many characteristics in common with the early stages of aortic stenosis. This opens up the potential of retarding the process through intervention strategies. SUMMARY: Hyperlipidaemia is associated with degenerative aortic valve stenosis, and the disease resembles the inflammatory process of atherosclerosis. Randomized controlled clinical trials will be needed to demonstrate the role of lipid intervention in patients with this condition.