Functional analysis of the 3′-terminal sequence of the maize controlling element (Ac) by internal replacement and deletion mutagenesis

Using deletion analysis of the Ac transposable element, we have shown that replacement of internal sequences from base pairs 181–3559 does not abolish transposition. We have done sequential deletion analysis of the 3'-end of the Ac element and found that deletion of the major transposase binding sites (AAACGG) abolishes transposition. But, surprisingly, we found a 3'-terminal deletion of the transposase binding sites which also contained a 71-bp internal sequence between base pairs 3559 and 3630 retained transposition ability. This 71-bp internal sequence did not have a transposase (ORFa) binding motif. These data suggest that two different domains may be involved in the minimal sequence necessary for transposition. Finally, we have identified functional prokaryotic promoter sequences and ARS sequences within the 5' and 3'-termini of Ac, but cannot ascribe any function to these sequences.     

Recruiting physicians to rural practice. Suggestions for success.

Medical school graduates from 1986 to 1988 and current residents in 12 family practice residency programs in the Northwest (N = 302) were surveyed to identify important factors in the recruitment process for their first postresidency placement. The study sought to compare the recruitment practices of rural communities and urban sites. Specific questions addressed in the study concerned sources of information about practice opportunities, stage in training when job search was initiated, factors related to unsuccessful site visits, and activities scheduled in the visit. Results indicated that referrals from faculty were the most valued source of information. Most job searches were initiated in the first 6 months of the third year in training. An unreceptive physician community and a reluctant spouse or partner were substantial problems for residents making site visits to rural communities. Rural sites tended to provide a broader mix of professional and personal activities during the visit.     

The influence of particle size and multiple apoprotein E-receptor interactions on the endocytic targeting of beta-VLDL in mouse peritoneal macrophages

Low density lipoprotein (LDL) and beta-very low density lipoprotein (beta-VLDL) are internalized by the same receptor in mouse peritoneal macrophages and yet their endocytic patterns differ; beta-VLDL is targeted to both widely distributed and perinuclear vesicles, whereas LDL is targeted almost entirely to perinuclear lysosomes. This endocytic divergence may have important metabolic consequences since beta-VLDL is catabolized slower than LDL and is a more potent stimulator of acyl-CoA/cholesterol acyl transferase (ACAT) than LDL. The goal of this study was to explore the determinants of beta-VLDL responsible for its pattern of endocytic targeting. Fluorescence microscopy experiments revealed that large, intestinally derived, apoprotein (Apo) E-rich beta-VLDL was targeted mostly to widely distributed vesicles, whereas small, hepatically derived beta-VLDL was targeted more centrally (like LDL). Furthermore, the large beta-VLDL had a higher ACAT-stimulatory potential than the smaller beta-VLDL. The basis for these differences was not due to fundamental differences in the means of uptake; both large and small beta-VLDL were internalized by receptor-mediated endocytosis (i.e., not phagocytosis) involving the interaction of Apo E of the beta-VLDL with the macrophage LDL receptor. However, large beta-VLDL was much more resistant to acid-mediated release from LDL receptors than small beta-VLDL. Furthermore, partial neutralization of the multiple Apo Es on these particles by immunotitration resulted in a more perinuclear endocytic pattern, a lower ACAT-stimulatory potential, and an increased sensitivity to acid-mediated receptor release. These data are consistent with the hypothesis that the interaction of the multivalent Apo Es of large beta-VLDL with multiple macrophage LDL receptors leads to a diminished or retarded release of the beta-VLDL from its receptor in the acidic sorting endosome which, in turn, may lead to the widely distributed endocytic pattern of large beta-VLDL. These findings may represent a physiologically relevant example of a previously described laboratory phenomenon whereby receptor cross-linking by multivalent ligands leads to a change in receptor targeting.     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.     

What is the best age to circumcise? A medical and ethical analysis

Circumcision is often claimed to be simpler, safer and more cost-effective when performed in the neonatal period as opposed to later in life, with a greater benefit-to-risk ratio. In the first part of this paper, we critically examine the evidence base for these claims, and find that it is not as robust as is commonly assumed. In the second part, we demonstrate that, even if one simply grants these claims for the sake of argument, it still does not follow that neonatal circumcision is ethically permissible absent urgent medical necessity. Based on a careful consideration of the relevant evidence, arguments and counterarguments, we conclude that medically unnecessary penile circumcision—like other medically unnecessary genital procedures, such as ‘cosmetic’ labiaplasty—should not be performed on individuals who are too young (or otherwise unable) to provide meaningful consent to the procedure.     

Effects of cis and trans Genetic Ancestry on Gene Expression in African Americans

Variation in gene expression is a fundamental aspect of human phenotypic variation. Several recent studies have analyzed gene expression levels in populations of different continental ancestry and reported population differences at a large number of genes. However, these differences could largely be due to non-genetic (e.g., environmental) effects. Here, we analyze gene expression levels in African American cell lines, which differ from previously analyzed cell lines in that individuals from this population inherit variable proportions of two continental ancestries. We first relate gene expression levels in individual African Americans to their genome-wide proportion of European ancestry. The results provide strong evidence of a genetic contribution to expression differences between European and African populations, validating previous findings. Second, we infer local ancestry (0, 1, or 2 European chromosomes) at each location in the genome and investigate the effects of ancestry proximal to the expressed gene (cis) versus ancestry elsewhere in the genome (trans). Both effects are highly significant, and we estimate that 12±3% of all heritable variation in human gene expression is due to cis variants.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.