The Bison: 1956

This digital object was funded in part through a grant from the Andrew W. Mellon Foundation. The digitalization of this object was part of a collaborative effort with the Washington Research Library Consortium and George Washington University.

Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity

Acetaminophen (APAP) is safe at therapeutic levels but causes hepatotoxicity via N-acetyl-p-benzoquinone imine-induced oxidative stress upon overdose. To determine the effect of human (h) pregnane X receptor (PXR) activation and CYP3A4 induction on APAP-induced hepatotoxicity, mice humanized for PXR and CYP3A4 (TgCYP3A4/hPXR) were treated with APAP and rifampicin. Human PXR activation and CYP3A4 induction enhanced APAP-induced hepatotoxicity as revealed by hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities elevated in serum, and hepatic necrosis after coadministration of rifampicin and APAP, compared with APAP administration alone. In contrast, hPXR mice, wild-type mice, and Pxr-null mice exhibited significantly lower ALT/AST levels compared with TgCYP3A4/hPXR mice after APAP administration. Toxicity was coincident with depletion of hepatic glutathione and increased production of hydrogen peroxide, suggesting increased oxidative stress upon hPXR activation. Moreover, mRNA analysis demonstrated that CYP3A4 and other PXR target genes were significantly induced by rifampicin treatment. Urinary metabolomic analysis indicated that cysteine-APAP and its metabolite S-(5-acetylamino-2-hydroxyphenyl)mercaptopyruvic acid were the major contributors to the toxic phenotype. Quantification of plasma APAP metabolites indicated that the APAP dimer formed coincident with increased oxidative stress. In addition, serum metabolomics revealed reduction of lysophosphatidylcholine in the APAP-treated groups. These findings demonstrated that human PXR is involved in regulation of APAP-induced toxicity through CYP3A4-mediated hepatic metabolism of APAP in the presence of PXR ligands.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

[Mumps epidemic in vaccinated children in West Switzerland]

Since 1991, 6 years after the recommendation of universal childhood vaccination against measles, mumps, and rubella (MMR triple vaccine), Switzerland is confronted with a large number of mumps cases affecting both vaccinated and unvaccinated children. Up to 80% of the children suffering from mumps between 1991 and 1995 had previously been vaccinated, the majority with the Rubini vaccine strain. On the basis of a case-control study including 102 patients and 92 controls from the same pediatric population, a study of the humoral immune-response following vaccination with the Rubini vaccine in 6 young adult volunteers, and two different genetic studies, we investigated the complex problem of large scale vaccine failure in Switzerland. We conclude that the recently reported large number of Swiss mumps cases was caused by at least four interacting factors: 1. A vaccine coverage of 90-95% at the age of 2 years is necessary to interrupt mumps wild virus circulation. The nationwide vaccine coverage in Switzerland of some 80% in 27-36 month-old children is too low. 2. Primary vaccine failures (absence of seroconversion or unprotective low levels of neutralizing antibodies), as well as secondary vaccine failures due to the rapid decline of antibodies to mumps virus in our volunteers and controls, seem to be frequent after vaccination with the Rubini strain. 3. Despite its reported Swiss origin, the Rubini strain does not belong to the mumps virus lineages recently circulating in this area but is closely related to American mumps virus strains. 4. Differences in protein structure between the vaccine strain and the circulating wild type strains, and in particular a different neutralization epitope in the hemagglutinin neuraminidase protein, may additionally contribute to the lack of protection in vaccinated individuals.

Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, alpha2PI1-8-VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated alpha2PI1-8-VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound alpha2PI1-8-VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of alpha2PI1-8-VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that alpha2PI1-8-VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by alpha2PI1-8-VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells.

Bovine colostrogenesis is distinguished by the specific transfer of IgG1 from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E2 ) and progesterone (P4 ) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta-2-Microglobulin (β2M). We hypothesized that steroid hormone treatments (E2 and P4 ) of bovine mammary epithelial cells in vitro would induce up-regulation of IgG1 transcytosis candidate gene mRNA expression suggesting involvement in IgG1 transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up-regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG1 transcytosis in bovine mammary cells.

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

Getting to Know You: Psychoeducational Groups to Counter Social Isolation of Neglectful Mothers

This research indicates a uniformly positive use of psychoeducational groups to counter social isolation of neglectful mothers. This research was supported by a National Child Welfare Fellowship from the U.S. Children 's Bureau to the author. The author thanks Nancy Dickinson, Sherrill Clark, and the staff of the California Social Work Education Center at the University of California for their oversight and guidance during (his fellowship. The author is also grateful to her fellow fellows for their input and guidance during this research effort. Special thanks to Rose Ben ham, Anna Bowen, Judith Brewington, Caron Byington, Scottye Cash. Dottie Dixon, and Verna Rickard for their support of this project.

Antarctic Glacial History Since the Last Glacial Maximum: An Overview of the Record on Land

This overview examines available circum-Antarctic glacial history archives on land, related to developments after the Last Glacial Maximum (LGM). It considers the glacial-stratigraphic and morphologic records and also biostratigraphical information from moss banks, lake sediments and penguin rookeries, with some reference to relevant glacial marine records. It is concluded that Holocene environmental development in Antarctica differed from that in the Northern Hemisphere. The initial deglaciation of the shelf areas surrounding Antarctica took place before 10000 C-14 yrs before present(sp), and was controlled by rising global sea level. This was followed by the deglaciation of some presently ice-free inner shelf and land areas between 10000 and 8000 yr sp. Continued deglaciation occurred gradually between 8000 yr sp and 5000 yr sp. Mid-Holocene glacial readvances are recorded from various sites around Antarctica. There are strong indications of a circum-Antarctic climate warmer than today 4700-2000 yr sp. The best dated records from the Antarctic Peninsula and coastal Victoria Land suggest climatic optimums there from 4000-3000 yr sp and 3600-2600 yr sp, respectively. Thereafter Neoglacial readvances are recorded. Relatively limited glacial expansions in Antarctica during the past few hundred years correlate with the Little Ice Age in the Northern Hemisphere.

Spatial Patterns of Intraseasonal Variability of Chlorophyll and Sea Surface Temperature in the California Current

Six years of daily satellite data are used to quantify and map intraseasonal variability of chlorophyll and sea surface temperature (SST) in the California Current. We define intraseasonal variability as temporal variation remaining after removal of interannual variability and stationary seasonal cycles. Semivariograms are used to quantify the temporal structure of residual time series. Empirical orthogonal function (EOF) analyses of semivariograms calculated across the region isolate dominant scales and corresponding spatial patterns of intraseasonal variability. The mode 1 EOFs for both chlorophyll and SST semivariograms indicate a dominant timescale of similar to 60 days. Spatial amplitudes and patterns of intraseasonal variance derived from mode 1 suggest dominant forcing of intraseasonal variability through distortion of large scale chlorophyll and SST gradients by mesoscale circulation. Intraseasonal SST variance is greatest off southern Baja and along southern Oregon and northern California. Chlorophyll variance is greatest over the shelf and slope, with elevated values closely confined to the Baja shelf and extending farthest from shore off California and the Pacific Northwest. Intraseasonal contributions to total SST variability are strongest near upwelling centers off southern Oregon and northern California, where seasonal contributions are weak. Intraseasonal variability accounts for the majority of total chlorophyll variance in most inshore areas save for southern Baja, where seasonal cycles dominate. Contributions of higher EOF modes to semivariogram structure indicate the degree to which intraseasonal variability is shifted to shorter timescales in certain areas. Comparisons of satellite-derived SST semivariograms to those calculated from co-located and concurrent buoy SST time series show similar features.

Cytochrome P450 Regulation by -Tocopherol in Pxr-Null and PXR-Humanized Mice

The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.