Identification of adenovirus (ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative ad (addl1520) for treatment of liver tumors

Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.     

Assessing overmedication: biology,philosophy and common sense

Overmedication is nowadays a serious problem in health care due to influences from the pharmaceutical industry and agencies responsible for regulation. The situation has indeed become appalling in psychiatry, where both theories and treatments have deteriorated under the impact of the industry. The overmedication problem is associated with biased biology in medicine. Adequate biological approaches would indicate that drug therapies must yield to diet therapies, particularly treatments involving omega-3 fatty acids, in many cases. To the extent that philosophy of science adapts to mainstream medicine in analyses of the current situation, it may reinforce the existing bias. To redress imbalances in health care, we ultimately have to rely on common sense.     

The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.     

A local influence approach applied to binary data from a psychiatric study

Recently, a lot of concern has been raised about assumptions needed in order to fit statistical models to incomplete multivariate and longitudinal data. In response, research efforts are being devoted to the development of tools that assess the sensitivity of such models to often strong but always, at least in part, unverifiable assumptions. Many efforts have been devoted to longitudinal data, primarily in the selection model context, although some researchers have expressed interest in the pattern-mixture setting as well. A promising tool, proposed by Verbeke et al. (2001, Biometrics 57, 43-50), is based on local influence (Cook, 1986, Journal of the Royal Statistical Society, Series B 48, 133-169). These authors considered the Diggle and Kenward (1994, Applied Statistics 43, 49-93) model, which is based on a selection model, integrating a linear mixed model for continuous outcomes with logistic regression for dropout. In this article, we show that a similar idea can be developed for multivariate and longitudinal binary data, subject to nonmonotone missingness. We focus on the model proposed by Baker, Rosenberger, and DerSimonian (1992, Statistics in Medicine 11, 643-657). The original model is first extended to allow for (possibly continuous) covariates, whereafter a local influence strategy is developed to support the model-building process. The model is able to deal with nonmonotone missingness but has some limitations as well, stemming from the conditional nature of the model parameters. Some analytical insight is provided into the behavior of the local influence graphs.     

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.     

Supplementation with vitamin E but not with vitamin C lowers lipid peroxidation in vivo in mildly hypercholesterolemic men.

Although the use of vitamin E supplements has been associated with a reduction in coronary events, assumed to be due to lowered lipid peroxidation, there are no previous long-term clinical trials into the effects of vitamin C or E supplementation on lipid peroxidation in vivo. Here, we have studied the long-term effects of vitamins C and E on plasma F2-isoprostanes, a widely used marker of lipid peroxidation in vivo. As a study cohort, a subset of the "Antioxidant Supplementation in Atherosclerosis Prevention" (ASAP) study was used. ASAP is a double-masked placebo-controlled randomized clinical trial to study the long-term effect of vitamin C (500 mg of slow release ascorbate daily), vitamin E (200 mg of D-alpha-tocopheryl acetate daily), both vitamins (CellaVie), or placebo on lipid peroxidation, atherosclerotic progression, blood pressure and myocardial infarction (n = 520 at baseline). Lipid peroxidation measurements were carried out in 100 consecutive men at entry and repeated at 12 months. The plasma F2-isoprostane concentration was lowered by 17.3% (95% CI 3.9-30.8%) in the vitamin E group (p = 0.006 for the change, as compared with the placebo group). On the contrary, vitamin C had no significant effect on plasma F2-isoprostanes as compared with the placebo group. There was also no interaction in the effect between these vitamins. In conclusion, long-term oral supplementation of clinically healthy, but hypercholesterolemic men, who have normal vitamin C and E levels with a reasonable dose of vitamin E lowers lipid peroxidation in vivo, but a relatively high dose of vitamin C does not. This observation may provide a mechanism for the observed ability of vitamin E supplements to prevent atherosclerosis.     

Floccular complex spike response to transparent retinal slip.

The flocculus of the rabbit is involved in the plasticity of compensatory eye movements. It is generally assumed that the climbing fiber input to floccular Purkinje cells encodes "retinal slip," which in turn would be a measure for the oculomotor performance error. To test this, we used transparent motion stimuli, creating a retinal slip signal that broke up this relation. We recorded the ensuing oculomotor behavior and complex spike activity of floccular Purkinje cells. Complex spike modulation in response to transparent stimulation was identical to that of a single optokinetic pattern, despite considerably different retinal slip. These results suggest that the climbing fiber code may be effectively related to the eye movement performance error, rather than to retinal slip.     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three hetero-oligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented.