Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Sporocarp ontogeny in Panus (Basidiomycotina): evolution and classification

Ontogenies of cultured Panus conchatus, P. rudis, and P. fulvus sporocarps were observed macroscopically and with scanning electron microscopy. Hymenophore differentiation in Panus involves periclinal growth of context hyphae below a closed surface palisade of hymenial elements, resulting in a cantharelloid appearance and radiate trama. This pattern is qualitatively different from that in Lentinus s. str., which suggests that lamellae of Panus and Lentinus are not homologous. Panus conchatus and P. rudis sporocarps have short stipes, develop directly from the mycelium, and mature in 5–10 d. Panus fulvus sporocarps have an elongate stipe, develop from a pseudosclerotium, and mature in about 3 wk, the first approximately 15 d of which involve apical elongation of a stipelike primordium that is able to dedifferentiate and regenerate cut apices. Panus conchatus and P. rudis sporocarps lacked regeneration ability. Panus conchatus sporocarps developed an ephemeral partial veil that was obliterated during sporocarp expansion. Outgroup comparison suggests that evolutionary changes in developmental programs in Panus have included: 1) delay in offset of primordium growth, with a corresponding increase in primordium size and time to maturation (hypermorphosis); 2) insertion of the pseudosclerotial stage in ontogeny; 3) gain of ability for dedifferentiation and regeneration; and 4) nonterminal gain or loss of veil tissue.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.