Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

Genomic imprinting in testicular germ cell tumours

Genomic imprinting refers to the parental origin-specific functional difference between the paternally and maternally-derived mammalian haploid genome. Normal embryogenesis depends on the presence of both a paternal and a maternal copy of particular chromosomal regions, containing the so-called imprinted genes. Genomic imprinting is established somewhere in the maturation from a primordial germ cell to a mature gamete, either spermatid or oocyte. We discuss the value of testicular cancers, especially those derived from the germ cell lineage, as a model to study erasement of the biparental pattern of genomic imprinting as present in the zygote and establishment of the paternal pattern during spermatogenesis. In addition, we will present data on the presence of X-inactivation in these cancers.     

Molecules to maps: tools for visualization and interaction in support of computational biology

The volume of data produced by genome projects, X-ray crystallography, NMR spectroscopy, and electron and confocal microscopy present the bioinformatics community with new challenges for analyzing, understanding, and exchanging this data. At the 1998 Pacific Symposium on Biocomputing, a track entitled 'Molecules to Maps: Tools for Visualization and Interaction in Computational Biology' provided tool developers and users with the opportunity to discuss advances in tools and techniques to assist scientists in evaluating, absorbing, navigating, and correlating this sea of information, through visualization and user interaction. In this paper we present these advances and discuss some of the challenges that remain to be solved.     

Effects of exogenous recombinant ovine interferon tau on circulating concentrations of progesterone, cortisol, luteinizing hormone, and antiviral activity; interestrous interval; rectal temperature; and uterine response to oxytocin in cyclic ewes

Interferon tau (IFN tau) is the conceptus-produced antiluteolytic signal in ruminants. Three experiments examined the effects of s.c. administration of recombinant ovine (ro)IFN tau on interestrous interval (IEI), oxytocin (OT)-induced uterine prostaglandin F2alpha metabolite (PGFM) production, rectal temperature (RT), respiration rate (RR), and plasma concentrations of progesterone, cortisol, LH, and antiviral activity (AVA) in plasma and uterine flushings. In experiment I, 20 ewes were treated s.c. with either 0, 1, 2, or 4 mg/day roIFN tau (0.7 x 10(8) U/mg; 5 ewes/dosage) from Days 11 to 15 of the estrous cycle (estrus = Day 0) and were challenged with OT (30 IU) on Day 15. Jugular blood samples were collected at -10, 0, 10, 20, 30, 40, 50, and 60 min relative to the OT challenge and assayed for PGFM. Recombinant oIFN tau increased IEI (16.7, 18.7, and 22.6 +/- 0.6 days for 0, 2, and 4 mg roIFN tau, respectively, p < 0.01). Recombinant oIFN tau did not affect peak PGFM response to OT (2309 +/- 172 pg/ml; p > 0.1). However, the 4 mg/day dosage delayed the time to peak PGFM (32.4 vs. 47.5 +/- 3.4 min; p < 0.01, 0 vs. 4 mg) and resulted in approximately 200% higher concentrations of PGFM at 60 min post-OT (0 vs. 4 mg/day, p < 0.07). Experiment II was similar to experiment I, except that only the 0- and 4-mg/day dosages of roIFN tau were administered. Ewes were hysterectomized on Day 16, and assay of uterine flushes detected no AVA from ewes treated with either 0 or 4 mg/day roIFN tau. In experiment III, 20 ewes were treated s.c. with either 0, 2, 4, or 6 mg roIFN tau on Day 12. Blood samples, RT, and RR were obtained at frequent intervals for 24 h, and plasma was assayed for progesterone, cortisol, LH, and AVA. Plasma AVA, which increased in a dose-dependent manner, was detectable within 60 min and remained elevated at 24 h compared to control values. RT (elevated 0.5-1.0 degrees C), RR, and cortisol increased in response to all dosages of roIFN tau, with peak values occurring 150-180 min postinjection. For all dosages of roIFN tau, plasma progesterone declined from 120 to 360 min posttreatment and then returned to pretreatment values by 24 h (p < 0.01) as compared to controls. Overall, exogenous roIFN tau altered uterine PGFM response to OT from a pulse to a gradual and sustained elevation and extended IEI with only a transient decline in progesterone and mild hyperthermia, effects that are not expected to compromise pregnancy.     

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.     

Six-month outcome of critically ill patients given glutamine-supplemented parenteral nutrition

Tetrahydrobiopterin is an essential cofactor required for activity of nitric oxide synthases. Existing evidence suggests that, during activation of constitutive and inducible isoforms of nitric oxide synthase, tetrahydrobiopterin is needed for allosteric and redox activation of enzymatic activity. However, precise mechanisms underlying the role of tetrahydrobiopterin in regulation of nitric oxide formation is not fully understood. In cerebral and peripheral arteries, increased availability of tetrahydrobiopterin can augment production of nitric oxide. In contrast, in arteries depleted of tetrahydrobiopterin, production of nitric oxide is impaired. Proinflammatory cytokines enhance mRNA expression of the rate-limiting enzyme of tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I and stimulate production of tetrahydrobiopterin. The ability of vascular tissues to synthesize tetrahydrobiopterin plays an important role in regulation of nitric oxide synthase under physiological conditions as well as during inflammation and sepsis. More recent studies concerning expression and function of recombinant nitric oxide synthases suggest that availability of tetrahydrobiopterin is important for production of nitric oxide in genetically engineered blood vessels. In this review, mechanisms regulating availability of intracellular tetrahydrobiopterin and its role in control of vascular tone under physiological and pathological conditions will be discussed.     

Forming limit diagrams calculated using Hill’s nonquadratic yield criterion

Forming limit diagrams (FLD) are calculated based on an extension of previous analyses by Jones and Gillis^[4,5,6] and Choiet al. ^[8,9] They idealized the sheet metal deformation into three phases: (I) homogeneous deformation up to maximum load, (II) deformation localization under constant load, and (III) local necking with a precipitous drop in load. They described the plastic behavior of sheet metals using a generalized quadratic flow law proposed by Jones and Gillis (JG). In the current analysis, Hill’s nonquadratic flow law for sheets having in-plane isotropy{su[101]} is used in conjunction with the three-stage deformation approximation. Calculated FLDs com- pare very favorably to experimentally determined results for aluminum-killed (AK) steel and aluminum alloys 2036-T4, 1100-H19, 5052-H32, 3003-0, and 3004-0. The comparison is fair for the titanium alloy Ti-6A1-4V. The agreement is poor for the strain rate-insensitive aluminum alloys 5052-0, 5052-H241, 5154-H111, and 6061-T4. Formerly with the University of Kentucky.