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.     

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.     

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.     

Effects of temperature, photoperiod, and light intensity on the calling rhythm in Arctiid moths

Mean times of onset for calling in Haploa clymene (Brown), Spilosoma virginica (Fabricius), Pareuchaetes insulata (Walker), Cycnia tenera (Hübner), and Euchaetes egle (Drury) advance to earlier times in the photoperiod at lower temperatures. Temperature has no apparent effect on the calling period in Pyrrharctia isabella (J. E. Smith), Spilosoma congrua Walker, and Apantesis nais (Drury). The relationship between the temperatures experienced by each of these species as adults and the response of their calling rhythms to temperature is discussed. Lights-on can elicit calling behaviour in C. tenera, although it is not an absolute requirement because calling eventually begins when lights-on is delayed 4 h and calling also begins prior to lights-on at lower temperatures. Calling periods lengthen in C. tenera and S. congrua when the scotophase is prolonged and in S. congrua after the onset of a lower photophase light intensity (40 lux), suggesting that a higher photophase light intensity (450 lux) inhibits calling and thus causes its termination.

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Résumé Aux faibles températures le moment moyen de déclenchement de l'appel apparaît plus tôt au cours de la photopériode chez Haploa clymene Brown, Spilosoma virginica Fab., Pareuchaetes insulata Walk., Cycnia tenera Hübn. et Euchaetes egle Drury. La température n'a apparemment pas d'effet sur le moment où l'appel débute chez Pyrrharctica isabella J.E. Smith, S. congrua Walk. et Apantesis nais Drury.L'analyse porte sur les relations entre les températures subies par les adultes de ces espèces et leurs réactions d'appel aux différentes températures. L'apparition de la lumière peut induire le comportement d'appel chez C. tenera, bien que ce ne soit pas indispensable puisqu'il peut éventuellement commencer à des températures plus basses avant l'illumination quand celle-ci est retardée de 4 heures. Les périodes d'appel sont prolongées avec la scotophase chez C. tenera et S. congrua, et même après l'apparition d'une photophase à faible intensité lumineuse (40 lux), l'appel de S. congrua se poursuit, ce qui suggère que les photophases à intensité lumineuse plus élevée (450 lux) inhibent l'appel et ainsi en provoquent la fin.

     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

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     

Helix geometry and hydration in an A-DNA tetramer: IC-C-G-G

The DNA oligomer of sequence IC-C-G-G has been synthesized, and its X-ray crystal structure solved at a resolution of 2.0 A, using anomalous scattering from iodines in phase analysis: 48 cycles of Jack-Levitt restrained least-squares refinement resulted in a residual error of 19.9% over all data, or 16.5% for two-sigma data. Two double-helical tetramers stack in the crystal to form a continuous octamer, except for the two missing phosphate connections across the center. The octamer has a mean helix rotation of 33.7 degrees (10.7 base-pairs per turn), rise of 2.87 A, mean inclination angle of base-pairs of 14 degrees, and mean base-pair propeller twist of +16.3 degrees. Local variations in both helix rotation and base plane roll angles, including those across the center of the octamer, are as predicted from base sequence by sum functions sigma 1 and sigma 2. The three known DNA octamers: IC-C-G-G/IC-C-G-G, G-G-T-A-T-A-C-C and G-G-C-C-G-G-C-C, make up a graded series in this order, with monotonically changing structural parameters. An exhaustive comparison of torsion angle correlations among the known A helices confirms some structural expectations and reveals some new features. 86 water molecules have been located per double-helical IC-C-G-G tetramer (the asymmetric unit), of which 451/2 per tetramer lie within a first hydrogen-bonded shell of hydration. No ordered water structure is observed comparable to the minor groove spine of hydration in B-DNA.     

Calling behaviour in arctiid moths: The effects of temperature and wind speed on the rhythmic exposure of the sex attractant gland

Female arctiid moths rhythmically extrude their ovipositors and thereby rhythmically expose their sex pheromone glands while calling. In Utetheisa ornatrix this behaviour results in concentration-modulated or pulsed pheromone release. The ovipositor protrusion frequencies were determined for 15 arctiid species: U. ornatrix, Haploa clymene, H. colona, H. lecontei, Pyrrarctia isabella, Estigmene acrea, Spilosoma congrua, S. virginica, Ecpantheria scribonia, Phragmatobia fuliginosa, Apantesis nais, A. arge, Pareuchaetes insulata, Cycnia tenera, and Euchaetes egle. Moth species that call early in the scotophase extrude their ovipositors at high frequencies (up to 170.9 ± 24.9 extrusions/min at 25°C) while moth species that call late in scotophase extrude their ovipositors at low frequencies (down to 68.3 ± 5.4 extrusions/min at 25°C). In all species tested, the ovipositor extrusion frequencies were shown to be temperature sensitive with a mean Q₁₀ of 2.0 ± 0.1. In one species, U. ornatrix, the ovipositor extrusion frequency varies with wind speed. In still air the ovipositor extrusion frequency is 70.1 ± 8.4 extrusions/min and at a wind speed of 120 cm/s the ovipositor extrusion frequency is 142.1 ± 8.3 extrusions/min. Suggested functions for rhythmic ovipositor extrusion during calling are discussed.     

Signatures of miR-181a on the Renal Transcriptome and Blood Pressure

MicroRNA-181a binds to the 3′ untranslated region of messenger RNA (mRNA) for renin, a rate-limiting enzyme of the renin-angiotensin system. Our objective was to determine whether this molecular interaction translates into a clinically meaningful effect on blood pressure and whether circulating miR-181a is a measurable proxy of blood pressure. In 200 human kidneys from the TRANScriptome of renaL humAn TissuE (TRANSLATE) study, renal miR-181a was the sole negative predictor of renin mRNA and a strong correlate of circulating miR-181a. Elevated miR-181a levels correlated positively with systolic and diastolic blood pressure in TRANSLATE, and this association was independent of circulating renin. The association between serum miR-181a and systolic blood pressure was replicated in 199 subjects from the Genetic Regulation of Arterial Pressure of Humans In the Community (GRAPHIC) study. Renal immunohistochemistry and in situ hybridization showed that colocalization of miR-181a and renin was most prominent in collecting ducts where renin is not released into the systemic circulation. Analysis of 69 human kidneys characterized by RNA sequencing revealed that miR-181a was associated with downregulation of four mitochondrial pathways and upregulation of 41 signaling cascades of adaptive immunity and inflammation. We conclude that renal miR-181a has pleiotropic effects on pathways relevant to blood pressure regulation and that circulating levels of miR-181a are both a measurable proxy of renal miR-181a expression and a novel biochemical correlate of blood pressure.