Preliminary X-ray study of crystals of human C-reactive protein

Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Protein Production and Crystallization at SECSG – An Overview

Using a high degree of automation, the Southeast Collaboratory for Structural Genomics (SECSG) has developed high throughput pipelines for protein production, and crystallization using a two-tiered approach. Primary, or tier-1, protein production focuses on producing proteins for members of large Pfam families that lack a representative structure in the Protein Data Bank. Target genomes are Pyrococcus furiosus and Caenorhabditis elegans. Selected human proteins are also under study. Tier-2 protein production, or target rescue, focuses on those tier-1 proteins, which either fail to crystallize or give poorly diffracting crystals. This two tier approach is more efficient since it allows the primary protein production groups to focus on the production of new targets while the tier-2 efforts focus on providing additional sample for further studies and modified protein for structure determination. Both efforts feed the SECSG high throughput crystallization pipeline, which is capable of screening over 40 proteins per week. Details of the various pipelines in use by the SECSG for protein production and crystallization, as well as some examples of target rescue are described.     

Crystallization and preliminary X-ray investigation of factor D of human complement

Human factor D, an essential enzyme of the alternative pathway of complement activation, has been crystallized. Crystals were grown by vapor diffusion using polyethylene glycol 6000 and NaCl as precipitants. The factor D crystals are triclinic and the space group is P1 with unit cell dimensions a = 40.8 A, b = 64.7 A, c = 40.3 A, alpha = 101.0 degrees, beta = 109.7 degrees, gamma = 74.3 degrees. The unit cell contains two molecules of factor D related by a non-crystallographic 2-fold axis. The crystals grow to dimensions of 0.8 mm x 0.5 mm x 0.2 mm within five days, are stable in the X-ray beam and diffract beyond 2.5 A.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

Background  

Uracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG.     

Relaxin-3 and RXFP3 expression, and steroidogenic actions in the ovary of teleost fish

Relaxins are peptides similar in secondary structure to insulins. In teleost genomes, five or six relaxin genes have been identified. Two relaxins group closely with mammalian relaxin-3 on phylogenetic analysis and are named relaxin-3a and b. We refer to the remainder as relaxins c to f. Ovarian expression of relaxin-3a, d and f genes, and the relaxin-3 receptor gene Rxfp3, was studied in Danio rerio using RT-PCR. Immunohistochemistry was used to determine the distribution of relaxin-3 peptides and RXFP3 in the ovary of Fundulus heteroclitus (killifish). Thirdly, enzyme immunoassays and ovarian follicular culture were used to determine the effect of treatment with human recombinant relaxin-3 on the production of 17beta-estradiol and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in killifish ovarian follicles. Relaxin-3a, d, f, and Rxfp3 genes were expressed regardless of sex or reproductive condition. Relaxin-3 immunostaining was present in mid to late follicular stages within cortical alveoli of the oocyte cytopasm, whereas receptor staining was localized to follicular cells. Treatment with relaxin-3 enhanced 17beta-estradiol production in early and late maturing follicles, but did not have an effect in vitellogenic follicles. Relaxin-3 appeared to suppress the release of MIS production. This suggests that relaxin peptides may be involved with estradiol-dependant events in follicular development.     

Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The ability to synthesize chiral building block molecules with high optical purity is of considerable importance to the fine chemical and pharmaceutical industries. Production of one such compound, 3-hydroxyvalerate (3HV), has previously been studied with respect to the in vivo or in vitro enzymatic depolymerization of biologically-derived co-polymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). However, production of this biopolymeric precursor typically necessitates the supplementation of a secondary carbon source (e.g., propionate) into the culture medium. In addition, previous approaches for producing 3HV have not focused on its enantiopure synthesis, and thus suffer from increased costs for product purification.     

Preservation of fresh edible cactus stems (<i>Opuntia ficus indica</i> Mill.) by modified atmosphere packaging

Cactus stems, the cladodes of Opuntia spp. cacti, are consumed in Mexico and other countries due to their fresh and herbaceous flavor, and because of their widely known nutraceutical benefits. In order to extend the postharvest life of this vegetable, the effect of a modified atmosphere packaging (MAP) was studied in cactus stems of the cultivar Atlixco stored at 4 ± 1 °C for 20 days under three types of atmospheres: (1) air (passive atmosphere), (2) 5 kPa O₂ + 4 kPa CO₂, and (3) N₂. During storage, the titratable acidity decreased and the color of cladodes became darker and less green; however, the 5 kPa O₂ + 4 kPa CO₂ atmosphere was able to preserve both quality characteristics. All modified atmospheres reduced weight loss (from 8 to <2%) and the symptoms of chilling injury, and this physiological disorder appeared earlier in controls than in MAP-stored cladodes. The levels of fermentation metabolites were low in all three evaluated atmospheres. Because of this, only cladodes stored under the N₂ atmosphere were selected for furthersensory analysis of the MAP effect on odor perception as evaluated by a trained panel. Results indicated that there was no detrimental effect (atypical odors) of MAP on this sensory characteristic. We conclude that cultivar Atlixco is suitable for preservation using MAP technology.     

Visualization of elusive structures using intracardiac echocardiography: Insights from electrophysiology

Echocardiography has the ability to noninvasively explore hemodynamic variables during pharmacologic or exercise stress test in patients with heart failure. In this review, we detail some important potential applications of stress echocardiography in patients with heart failure. In patients with coronary artery disease and chronic LV dysfunction, dobutamine stress echocardiography is able to distinguish between viable and fibrotic tissue to make adequate clinical decisions. Exercise testing, in combination with echocardiographic monitoring, is a method of obtaining accurate information in the assessment of functional capacity and prognosis. Functional mitral regurgitation is a common finding in patients with dilated and ischaemic cardiomyopathy and stress echocardiography in the form of exercise or pharmacologic protocols can be useful to evaluate the behaviour of mitral regurgitation. It is clinical useful to search the presence of contractile reserve in non ischemic dilated cardiomyopathy such as to screen or monitor the presence of latent myocardial dysfunction in patients who had exposure to cardiotoxic agents. Moreover, in patients with suspected diastolic heart failure and normal systolic function, exercise echocardiography could be able to demonstrate the existence of such dysfunction and determine that it is sufficient to limit exercise tolerance. Finally, in the aortic stenosis dobutamine echocardiography can distinguish severe from non-severe stenosis in patients with low transvalvular gradients and depressed left ventricular function.