Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

Aortic ascorbic acid, trace elements, and superoxide dismutase activity in human aneurysmal and occlusive disease

Altered trace elements and ascorbic acid metabolism have been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, their role in the disease process, or the effect of atherosclerosis on their tissue levels within plaque, is poorly understood. The present study analyzes the concentrations of Fe, Cu, Zn, and Mn, and ascorbic acid and superoxide dismutase (SOD) activity in tissue samples from 29 patients with abdominal aortic aneurysms (AAA) and 14 patients with atherosclerotic occlusive disease (AOD). It was observed that the Fe and Mn concentrations in AAA and AOD tissue were higher than the levels in nondiseased control aorta, whereas Cu and Zn levels in AAA and AOD tissue were similar to the levels in controls. The Zn:Cu ratio was significantly lower in the AAA tissue in comparison to both AOD and control tissue. In addition, AAA and AOD tissue had low ascorbic acid levels and low Cu,Zn-SOD activity with Cu,Zn-SOD:Mn-SOD ratios of 0.27 and 0.19, respectively, compared to a ratio of 3.20 in control aorta. These data indicate that aorta affected by aneurysms and occlusive disease have altered trace element and ascorbic acid concentrations, as well as low Cu,Zn-SOD activity. Although these observations do not directly support the hypothesis that AAA is associated with aortic Cu deficiency they do suggest a role for oxygen radicals or increased lipid peroxidation in occlusive and aneurysmal disease of the aorta.     

Molecular classification of scrapie strains in mice using gene expression profiling

Transmissible spongiform encephalopathy strains demonstrate specific prion characteristics, each with specific incubation times, and strain-specific patterns of deposition of the misfolded isoform of prion, PrPSc, in the brains of infected individuals. Different biochemical properties, including glycosylation profiles and the degree of proteinase resistance, have been shown to be strain-specific. However, no relationship between these properties and the phenotypic differences in the subsequent diseases has as yet been determined. Here we explore the utility of gene expression profiles to identify differences in the host response to different strains of prion agent. We identify 114 genes that exhibit significantly different levels of expression in mice infected with three strains of scrapie. These genes represent a pool of genes involved in a strain-specific response to prion disease. We have identified the most discriminatory genes from this list utilizing a wrapper-based feature selection algorithm with external cross-validation.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Voracity and prey preference of insidious flower bug (hemiptera: anthocoridae) for immature stages of soybean aphid (hemiptera: aphididae) and soybean thrips (thysanoptera: thripidae)

The generalist predator, Orius insidiosus (Say), is an important natural enemy of the soybean aphid, Aphis glycines Matsumura. Soybean thrips, Neohydatothrips variabilis (Beach), serve as an important prey resource for O. insidiosus in soybeans, sustaining the predator's population before the arrival of the soybean aphid. Although generalist predators can forage on a broad range of prey, they may show distinct preferences for particular prey, attacking prey at levels disproportionate to their relative numbers. To assess the preference of O. insidiosus for soybean aphid and soybean thrips, attack rates of nymphal and adult O. insidiosus were measured in the laboratory. For both adults and nymphs, the number of prey attacked increased as more prey were provided. For nymphs, the total number of prey attacked increased as the predator matured. In general, the number of prey attacked by adult predators was relatively constant as the predator aged. Both O. insidiosus nymphs and adults displayed a preference for soybean thrips, by disproportionately attacking soybean thrips over soybean aphid regardless of the relative densities of the two prey. We discuss implications of this preference on O. insidiosus life history characteristics and the potential impact on O. insidiosus-prey dynamics in the field.     

The emergence of Cochlodinium along the California Coast (USA)

A sudden and nearly synchronous emergence of the red tide forming dinoflagellate Cochlodinium along more than 800 km of California coastline was initially observed in late summer 2004. Thereafter high cell concentrations have been detected on an annual basis. Here, we present quantitative and semi-quantitative data indicating that Cochlodinium was uncommon in the phytoplankton community in California prior to 2004 and is now persisting as a more regular component and one that seasonally can cause red tides. The quantitative portion of this study was primarily conducted in Monterey Bay, where cell densities reached at least 6 × 10⁴ cells L⁻¹ during the initial outbreak. A semi-quantitative comparison of California coastal counties by the California Department of Health Services (CDHS) was also made: of the 15 counties surveyed (most with multiple sites per county), cells were detected only from Los Angeles County in the south to San Mateo County in the central region (seven counties), but not in the northern part of the state (six counties). Two counties in the central region of the state, San Luis Obispo and Santa Cruz, displayed intense and frequent periods of elevated Cochlodinium cell abundances. Although not observed in the state-wide CDHS survey, we occasionally found cells in San Diego County with densities up to 2.7 × 10⁴ cells L⁻¹. Though these colonial dinoflagellates have been recognized in California for over 80 years, with several “blooms” recorded prior to 2004, the species’ geographic range and abundance in recent years suggest significant shifts in the nearshore phytoplankton community of this region of the eastern Pacific.