Effect of fully sulfated glycosaminoglycans on pulmonary artery smooth muscle cell proliferation.

Fully sulfated heparin and other glycosaminoglycans, namely heparan, chondroitin, and dermatan sulfates, and hyaluronan have been prepared by using sulfur trioxide under mild chemical conditions. All these derivatives were assayed for antiproliferative activity on cultured bovine pulmonary artery smooth muscle cells (BPASMCs). No appreciable difference was found between heparin and fully sulfated heparin. Chondroitin and dermatan sulfates actually stimulated BPASMCs growth but full sulfonation made them strongly antiproliferative. Native hyaluronan was not antiproliferative but became strongly so after sulfonation. Neither acharan sulfate nor N-sulfoacharan sulfate had any antiproliferative activity. This suggests that O-sulfonation of the polysaccharide is critical for antiproliferative activity, whereas N-sulfonation of glucosamine residues is not.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

The isolation and characterization of mutants of the integration host factor (IHF) of Escherichia coli with altered, expanded DNA-binding specificities.

The integration host factor (IHF) of Escherichia coli is a small, basic protein that is required for lambda site-specific recombination and a variety of cellular processes. It is composed of two subunits, alpha and beta, that are encoded by the himA and hip (himD) genes, respectively. IHF is a sequence-specific DNA-binding protein and bends the DNA when it binds. We have used the bacteriophage P22-based challenge phage selection to isolate suppressor mutants with altered, expanded DNA binding specificities. The suppressors were isolated by selecting mutants that recognize variants of the phage lambda H'IHF recognition site. Two of the mutants recognize both the wild-type and a single variant site and contain amino acid substitutions at positions 64 (Pro to Leu) or 65 (Lys to Ser) of the alpha subunit. These substitutions are in a region of the protein that is predicted to contain a flexible arm that interacts with DNA. Three other mutants, which recognize the wild-type and a different variant site, contain amino acid substitutions at position 44 (Glu to Lys, Val or Gly) of the beta subunit. These substitutions are in the middle of a predicted beta-strand of the subunit. We discuss the possible mechanisms of suppression by the mutants in terms of a model of the IHF-DNA complex proposed by Yang and Nash [Cell, 57, 869-880 (1989)].     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

Structural models of Cys-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine receptor infer the existence of cytoplasmic portals within the conduction pathway framed by helical amphipathic regions (termed membrane-associated (MA) helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (Arg⁴³⁶ and Arg⁴⁴⁰) within the MA helix of 5-hydroxytryptamine type 3A (5-HT₃A) receptors act singularly as rate-limiting determinants of single-channel conductance (γ). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT₃A receptor model. We probed the majority of residues within the MA helix of the human 5-HT₃A subunit using alanine- and arginine-scanning mutagenesis and the substituted cysteine accessibility method to determine their relative influences upon γ. Numerous residues, prominently those at the 435, 436, 439, and 440 positions, were found to markedly influence γ. This approach yielded a functional map of the 5-HT₃A receptor portals, which agrees well with the homology model.     

The chromosomes of Schoutedenia lutea (homoptera,aphidoidea, greenideinae), with an account of meiosis in the male

Somatic chromosomes of both sexes and chromosome behaviour during spermatogenesis were studied in the aphid Schoutedenia lutea (van der Goot). Four long but unequal chromosomes in females were interpreted as X chromosomes (X₁X₁X₂X₂) with one member of an autosome pair attached to one X₁, and the other member to one X₂, so that the four long chromosomes were actually X₁+A, X₁, X₂+A, X₂. Males (normally XO in aphids) received X chromosomes corresponding in relative length to the two longest (X₁+A, X₂+A) in females. During spermatogenesis parallel pairing occurred in prophase 1 and the X₁ and X₂ chromosomes became associated via their autosomal segments. In anaphase I, the autosomal segment became detached from one of the X chromosomes and entered the non-viable (non-X-bearing) spermatocyte, while the viable spermatocyte received both X₁ and X₂ (either one of which still carried an autosome) and the haploid set of free autosomes. The consequences for sex determination and zygote formation of this unusual system are discussed; a stable chromosomal constitution for the zygote can be achieved only at the expense of considerable gamete wastage.     

GENETIC EVIDENCE FOR THE OCCURRENCE OF A NEW SPECIES OF NEOPHYLLAPHIS TAKAHASHI (HOMOPTERA: APHIDIDAE) IN AUSTRALIA

Abstract
Chromosomal and electrophoretic evidence is presented demonstrating the occurrence of a fourth species of Neophyllaphis Takahashi in Australia. The new species, N. lanata sp. n., occurs on Podocarpus spinulosus. Karyotypes of all four species of Neophyllaphis in Australia are provided.     

Analysis of adenovirus type 2 L1 RNA 3''-end formation in vivo and in vitro.

Downstream sequence requirements for efficient cleavage and polyadenylation at the adenovirus type 2 L1 poly(A) site were determined in vivo in 293 cells and in vitro by using RNA precursors in HeLa cell nuclear extracts. The two cleavage sites used were found to differ in sensitivity to 3'-end deletion in vivo and in vitro.