Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA.

Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo.     

Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1.

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.     

High-affinity NGF binding requires coexpression of the trk proto-oncogene and the low-affinity NGF receptor.

Nerve growth factor (NGF) interacts with two different low-affinity receptors that can be distinguished by affinity crosslinking. Reconstitution experiments by membrane fusion and transient transfection into heterologous cells indicate that high-affinity NGF binding requires coexpression and binding to both the low-affinity NGF receptor and the tyrosine kinase trk gene product. These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation involving trk and the low-affinity NGF receptor.     

Running energetics in the pronghorn antelope

The pronghorn antelope (Antilocapra americana) has an alleged top speed of 100 km h-1, second only to the cheetah (Acionyx jubatus) among land vertebrates, a possible response to predation in the exposed habitat of the North American prairie. Unlike cheetahs, however, pronghorn antelope are distance runners rather than sprinters, and can run 11 km in 10 min, an average speed of 65 km h-1. We measured maximum oxygen uptake in pronghorn antelope to distinguish between two potential explanations for this ability: either they have evolved a uniquely high muscular efficiency (low cost of transport) or they can supply oxygen to the muscles at unusually high levels. Because the cost of transport (energy per unit distance covered per unit body mass) varies as a predictable function of body mass among terrestrial vertebrates, we can calculate the predicted cost to maintain speeds of 65 and 100 km h-1 in an average 32-kg animal. The resulting range of predicted values, 3.2-5.1 ml O2 kg-1 s-1, far surpasses the predicted maximum aerobic capacity of a 32-kg mammal (1.5 ml O2 kg-1 s-1). We conclude that their performance is achieved by an extraordinary capacity to consume and process enough oxygen to support a predicted running speed greater than 20 ms-1 (70 km h-1), attained without unique respiratory-system structures.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein beta-adaptin

Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.     

New use of BCG for recombinant vaccines

BCG, a live attenuated tubercle bacillus, is the most widely used vaccine in the world and is also a useful vaccine vehicle for delivering protective antigens of multiple pathogens. Extrachromosomal and integrative expression vectors carrying the regulatory sequences for major BCG heat-shock proteins have been developed to allow expression of foreign antigens in BCG. These recombinant BCG strains can elicit long-lasting humoral and cellular immune responses to foreign antigens in mice.