Different postsynaptic events in two types of retinal bipolar cell

The first synapse in the vertebrate visual system is made between the photoreceptors and the biopolar cells. Bioplar cells fall into two distinct classes according to whether the cell hyperpolarizes or depolarizes to small centred spots of light. Most evidence indicates that the light-induced hyperpolarization of the photoreceptprs suppresses transmitter release from the synaptic terminals, and it is probable that the differences between the two bipolar cell classes results from the different actions of the photoreceptor transmitter. In analysing the membrane potential fluctuations in both types of bipolar cell we find that the voltage noise spectra differ. It is to be expected that postsynaptic noise would be composed of the sum of noise generated in and transmitted from the cones and the noise arising from the statistical nature of synaptic transmission. We report here evidence for two such components in the voltage noise spectra recorded from each type of bipolar cell. The differences in the frequency distribution of the presumed transmitter-related components indicates that the transmitter generates events of longer duration in the depolarizing biopolar cells.     

Amplification of immunoglobulin lambda constant genes in populations of wild mice

The lambda immunoglobulin light chain (Ig lambda) locus of BALB/c inbred mice consists of two variable region gene segments (V lambda)1-3, and four constant region gene segments (C lambda)1,2,4,5. Each C lambda gene segment is associated with a unique joining segment (J lambda)2,4-7, and they are organized in two paired units, J3C3-J1C1 and J2C2-J4C4 (refs 4, 8). Using cDNA probes specific for C lambda 1 and C lambda 2 (ref. 9) we have analysed the genomic organization of the C lambda gene segments in wild-derived and inbred strains of mice. Although Southern blots of the genomic DNA of inbred mice show a constant pattern of hybridization, wild-derived mice show a high degree of variation in the number, size and intensity of hybridizing fragments. We have now found that, per haploid genome, mice of a Mus musculus musculus stock isolated from Sladeckovce, Czechoslovakia (CzII) have at least 12 C lambda segments, and mice of a Mus musculus domesticus stock 'Centreville Lights' from Centreville, Maryland (CL) have at least 8 C lambda segments. There appears to have been relatively recent amplifications of the C lambda gene segments in wild mice.     

Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel

Many excitable cells contain at least two different voltage-dependent Ca channels (L- and T-type). The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation, which increases the probability of it being open, and is readily blocked by Ca channel blockers including dihydropyridines and phenylalkylamines. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo, that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules contains three peptides of relative molecular mass (Mr) 142,000 (142K), 56K and 31K. The cAMP kinase incorporates one mol phosphate per mol of the 142K peptide and binding of (+)PN-200/110, a potent Ca antagonist, is allosterically affected by D-cis-diltiazem and verapamil. The purified dihydropyridine-receptor complex has also been incorporated into phospholipid bilayer membranes. Here, we show for the first time that the complex can be reconstituted to form a functional 20-pS Ca channel that retains the principal regulatory, biochemical and pharmacological properties of membrane-bound L-type Ca channels.     

A malaria T-cell epitope recognized in association with most mouse and human MHC class II molecules

An ideal vaccine should elicit a long lasting immune response against the natural parasite, both at the T- and B-cell level. The immune response should occur in all individuals and be directed against determinants that do not vary in the natural parasite population. A major problem in designing synthetic peptide vaccines is that T cells generally recognize peptide antigens only in association with one or a few of the many variants of major histocompatibility complex (MHC) antigens. During the characterization of epitopes of the malaria parasite Plasmodium falciparum that are recognized by human T cells, we analysed a sequence of the circumsporozoite protein, and found that synthetic peptides corresponding to this sequence are recognized by T cells in association with many different MHC class II molecules, both in mouse and in man. This region of the circumsporozoite protein is invariant in different parasite isolates. Peptides derived from this region should be capable of inducing T-cell responses in individuals of most HLA-DR types, and may represent good candidates for inclusion in an effective anti-malaria peptide vaccine.     

Thymic selection process induced by hybrid antibodies

Thymus-derived (T) lymphocytes using the alpha beta T-cell antigen receptor (TCR) recognize fragmented antigen in conjunction with surface molecules encoded by genes of the major histocompatibility complex (MHC). Peripheral T lymphocytes preferentially see antigen presented by self rather than by foreign MHC molecules, and autoreactive T lymphocytes are deleted. Thus, the peripheral T-lymphocyte repertoire is skewed towards recognition of antigen in the context of self-MHC and towards tolerance to self-antigens. During T-lymphocyte development in the thymus, this repertoire is formed by the interaction of TCR with MHC molecules resulting in positive and negative selection phenomena. Hybrid antibodies (HAbs) that carry binding sites to the TCR and to a surface marker on another cell can engage all T lymphocytes regardless of their specificity. It should be possible to mimic selection processes in normal animals with HAb that specifically link members of a TCR family to MHC molecules on the thymic stroma. We have probed T-lymphocyte development with HAbs linking V beta 8-positive TCR to either class I or class II MHC products in thymic organ culture. Thymocytes exposed to either HAb in an early stage of maturation respond with a significant increase in the frequency of V beta 8-carrying cells. At a later stage of development V beta 8-positive thymocytes are depleted. These results illustrate the succession of positive and negative selection in the developing thymus of normal mice.     

Alternative splicing directs the expression of two D2 dopamine receptor isoforms

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological properties and the intracellular responses they mediate. The cerebral D2 dopamine receptor is the target of drugs used to alleviate the main symptoms of schizophrenia. Although it is considered to be a single molecular entity, there is evidence that multiple D2-receptor subtypes exist. A complementary DNA encoding a D2 receptor has recently been cloned and the deduced 415-amino-acid sequence indicates that it belongs to the large superfamily of receptors coupled to G proteins, and that its topology consists of seven transmembrane domains. In this family, the genes are frequently without introns and each is believed to encode a unique polypeptide product. Here we show that the gene for the D2 receptor produces two receptor isoforms by alternative messenger RNA splicing, providing a route to receptor diversity in this family. One isoform corresponds to the D2(415) receptor, but the second contains an additional sequence encoding a 29-amino-acid fragment, defining a novel D2(444) receptor isoform. Expression of the two isoforms is tissue-specific, and both are regulated by guanyl nucleotides. As the extra sequence is located within a putative cytoplasmic loop that binds to G proteins, the two isoforms might interact with different G proteins and thereby initiate distinct intracellular signals.     

T-cell receptor delta-chain can substitute for alpha to form a beta delta heterodimer

Specific monoclonal antibodies have made possible the identification of two T-cell antigen receptor (TCR) heterodimers, alpha beta TCR and gamma delta TCR. Formation of these receptors is largely separated by the preferential pairing of alpha-TCR with beta and gamma-TCR with delta, the sequential rearrangement and expression of the TCR loci during thymic development and the deletion of the delta-loci either prior to or concomitant with alpha-rearrangement in alpha beta TCR cells. Here we show that delta-TCR can substitute for alpha in pairing with beta to form a beta delta heterodimer. This receptor is expressed on the cell surface of the T-leukaemia cell line DND41 as analysed with beta- and delta-specific monoclonal antibodies. We suggest that a variety of factors including, for example, the deletion of the delta-TCR loci, can now be understood as exclusion mechanisms operating to prevent not only the formation of gamma delta receptors, but also of beta delta T-cell receptors, thereby promoting the numerically dominant alpha beta TCR lineage. Nevertheless, some developing T-cells that do not rearrange the alpha-loci may express the beta delta TCR as described here.     

Substrate specificity and affinity of a protein modulated by bound water molecules

Water molecules influence molecular interactions in all biological systems, yet it is extremely difficult to understand their effects in precise atomic detail. Here we present evidence, based on highly refined atomic structures of the complexes of the L-arabinose-binding protein with L-arabinose, D-fucose and D-galactose, that bound water molecules, coupled with localized conformational changes, can govern substrate specificity and affinity. The atoms common to the three sugars are identically positioned in the binding site and the same nine strong hydrogen bonds are formed in all three complexes. Two hydrogen-bonded water molecules in the site contribute further to tight binding of L-arabinose but create an unfavourable interaction with the methyl group of D-fucose. Equally tight binding of D-galactose is attained by the replacement of one of the hydrogen-bonded water molecules by its--CH2OH group, coordinated with localized structural changes which include a shift and redirection of the hydrogen-bonding interactions of the other water molecule. These observations illustrate how ordered water molecules can contribute directly to the properties of proteins by influencing their interaction with ligands.