Differential requirements for AP-2 in clathrin-mediated endocytosis

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.     

Patterns and mechanisms of selection on a family-diagnostic trait: evidence from experimental manipulation and lifetime fitness selection gradients

Plant traits that show little variation across higher taxa are often used as diagnostic traits, but the reason for the stasis of such traits remains unclear. Wild radish, Raphanus raphanistrum, exhibits tetradynamous stamens (four long and two short, producing a dimorphism in anther height within each flower), as do the vast majority of the more than 3,000 species in the Brassicaceae. Here we examine the hypothesis that selection maintains the stasis of dimorphic anther height by investigating the effects of this trait on pollen removal, seed siring success, and seed set in R. raphanistrum using both experimental and observational methods. Observational selection gradient analysis based on lifetime seed siring success provided evidence for an optimum dimorphism that was greater than zero in one of three years. In both experimentally manipulated and unmanipulated flowers, more pollen was removed in single visits from flowers with less dimorphism. There was no significant effect of anther dimorphism on female fitness (seed set). Therefore, there is some evidence to suggest that selection is maintaining anther dimorphism in wild radish, and that higher male fitness might result from restriction of single-visit pollen removal. We discuss these results in light of pollen presentation theory.     

Interaction of calcitonin-gene-related peptide with its receptors

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28-37 and 8-18 are normally required for high-affinity binding, while residues 19-27 form a hinge region. The 28-37 region is almost certainly in direct contact with the receptor; 8-18 may make additional receptor contacts or may stabilize an appropriate conformation of 28-37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.     

A prospective analysis of patients undergoing silicone breast implant explantation

Despite the lack of a scientifically proven link between silicone implants and disease, many women have chosen to have their implants removed out of concern for their health. Unfortunately, there are few studies in the literature that have investigated the outcome of explanations, and there are no prospective analyses of the effect explantation has on a patient's general health. The goal of this study was to use a prospective database to determine whether there were any preoperative parameters that could be used to predict which patients would be improved following removal of silicone breast implants and to provide a quantifiable measure of that improvement. A total of 38 patients with silicone breast implants underwent operative removal of their breast implants by faculty at the University of Texas Southwestern Medical Center. They were given questionnaires regarding several personal and medical parameters to be completed preoperatively, at 6 weeks postoperatively, and at 6 months postoperatively. In addition, their physicians completed preoperative and postoperative evaluations of the patient's general health status. A control group of 38 patients was established; they were matched with the experimental group with regard to age and other initial parameters. Their responses to questionnaires were then grouped according to standard subscales to evaluate physical functioning, physical role, bodily pain, general health, vitality, social functioning, emotional role, mental health, appearance evaluation, appearance orientation, illness orientation, and body area satisfaction. When compared with the control group, we found that patients who had undergone explantation showed a temporary decrease in musculoskeletal symptoms and bodily pain, as well as an increase in vitality, mental health, and body area satisfaction. Of the experimental group, those who initially indicated a higher number of musculoskeletal symptoms and a higher appearance evaluation were more likely to indicate a significant improvement in general health since explantation.     

Rotavirus 2/6 Viruslike Particles Administered Intranasally with Cholera Toxin, Escherichia coli Heat-Labile Toxin (LT), and LT-R192G Induce Protection from Rotavirus Challenge

We have shown that rotavirus 2/6 viruslike particles composed of proteins VP2 and VP6 (2/6-VLPs) administered to mice intranasally with cholera toxin (CT) induced protection from rotavirus challenge, as measured by virus shedding. Since it is unclear if CT will be approved for human use, we evaluated the adjuvanticity of Escherichia coli heat-labile toxin (LT) and LT-R192G. Mice were inoculated intranasally with 10 μg of 2/6-VLPs combined with CT, LT, or LT-R192G. All three adjuvants induced equivalent geometric mean titers of rotavirus-specific serum antibody and intestinal immunoglobulin G (IgG). Mice inoculated with 2/6-VLPs with LT produced significantly higher titers of intestinal IgA than mice given CT as the adjuvant. All mice inoculated with 2/6-VLPs mixed with LT and LT-R192G were totally protected (100%) from rotavirus challenge, while mice inoculated with 2/6-VLPs mixed with CT showed a mean 91% protection from challenge. The availability of a safe, effective mucosal adjuvant such as LT-R192G will increase the practicality of administering recombinant vaccines mucosally.     

A Farnesyltransferase Inhibitor Induces Tumor Regression in Transgenic Mice Harboring Multiple Oncogenic Mutations by Mediating Alterations in Both Cell Cycle Control and Apoptosis

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)–v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV–v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV–v-Ha-ras mice were interbred with p53^−/− mice. Tumors in ras/p53^−/− mice treated with L-744,832 regressed as efficiently as MMTV–v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G₁ with a corresponding decrease in the S-phase fraction. MMTV–v-Ha-ras mice were also interbred with MMTV–c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV–v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV–c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.     

Electrical energy production from forest detritus in a forested wetland using microbial fuel cells

Microbial fuel cell (MFC) technology has shown great potential for harvesting energy from waste organic materials. Here, we explored the potential of MFC‐based electricity generation from forest detritus, a large untapped biomass pool. Electricity generation from in situ MFCs and relevant environmental parameters (i.e., carbon sources and concentrations, temperature, water depth) in a seasonally flooded freshwater cypress‐tupelo wetland were monitored intensively for two flooding periods. Current outputs ranged from 0 to 1.27 mA (mean of 0.40 mA for flooding period) and were highly sensitive to environmental changes, showing seasonal and diel dependences. Excluding the influence of heavy storms, drought, or wetland icing, current output was highly temperature‐dependent dielly. Seasonally, current output gradually increased in the first 3–4 months (limited by temperature) and decreased slightly during the last 1–2 months (probably limited by carbon and nutrients) of both flooding periods. Litter extract of baldcypress (Taxodium distictum) with lower C/N ratio and aromatic content showed greater stable current outputs (0.57 mA) based on 50 mg l^?1 biological oxygen demand compared to extracts of water tupelo (Nyssa aquatica) and longleaf pine (Pinus palustris), suggesting that the current output of in situ MFCs could depend on the vegetation within a wetland. Our study highlights the potential application of MFC in generating green and sustainable electricity from forest biomass for powering remote sensors in wetland ecosystems.     

Biopharmaceutics Classification System-Based Biowaivers for Generic Oncology Drug Products: Case Studies

Establishing bioequivalence (BE) of drugs indicated to treat cancer poses special challenges. For ethical reasons, often, the studies need to be conducted in cancer patients rather than in healthy volunteers, especially when the drug is cytotoxic. The Biopharmaceutics Classification System (BCS) introduced by Amidon (1) and adopted by the FDA, presents opportunities to avoid conducting the bioequivalence studies in humans. This paper analyzes the application of the BCS approach by the generic pharmaceutical industry and the FDA to oncology drug products. To date, the FDA has granted BCS-based biowaivers for several drug products involving at least four different drug substances, used to treat cancer. Compared to in vivo BE studies, development of data to justify BCS waivers is considered somewhat easier, faster, and more cost effective. However, the FDA experience shows that the approval times for applications containing in vitro studies to support the BCS-based biowaivers are often as long as the applications containing in vivo BE studies, primarily because of inadequate information in the submissions. This paper deliberates some common causes for the delays in the approval of applications requesting BCS-based biowaivers for oncology drug products. Scientific considerations of conducting a non-BCS-based in vivo BE study for generic oncology drug products are also discussed. It is hoped that the information provided in our study would help the applicants to improve the quality of ANDA submissions in the future.KEY WORDS: Biopharmaceutics Classification System, bioequivalence, biowaiver, cancer, oncology