The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.     

Interaction of albumin with the endothelial cell surface

Endothelial cells (EC) are covered with cell-borne proteoglycans and glycoproteins. Blood plasma proteins (e.g., albumin) adsorb to this glycocalyx forming a complex endothelial surface layer (ESL). We determined the molecular mobility of albumin by electron spin resonance (ESR) in the presence and absence of ECs to analyze interactions with the ESL. Albumin was spin labeled with 5- or 12-4,4-dimethyloxazolidine-N-oxyl (DOXYL)-stearic acid yielding information on the mobility of the molecular surface (5-DOXYL) or the entire protein (12-DOXYL). EC cultures grown on glass coverslips were immersed in labeled albumin and placed in the temperature-regulated cavity of an ESR spectrometer. Alternatively, ECs were labeled and then exposed to native albumin. At 37 degrees C, rotational correlation times determined by modified saturation transfer ESR (ST-ESR) were 26 and 48 ns for 5-DOXYL- and 12-DOXYL-labeled albumin, respectively. Presence of ECs increased rotational correlation time values for 5-DOXYL-stearic acid to 37 ns but not for 12-DOXYL-stearic acid. Albumin was able to completely take up the label from labeled EC within 2 min. The present study shows that modified ST-ESR can be used to determine the mobility of biological macromolecules interacting with cellular surfaces. Reduction in albumin surface mobility in the presence of EC at unchanged mobility of protein proper and fast removal of labeled fatty acids from EC membranes indicate rapid transient interactions between albumin surface and ESL but no rigid incorporation of albumin into a macromolecular network that would interfere with its transport function for poorly water-soluble substances.     

Localization and identification of Schistosoma mansoni/KLH-crossreactive components in infected mice.

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

Regional distribution of glucose utilization in the telencephalon of toads in response to configurational visual stimuli: a14C-2DG study

Summary The regional glucose utilization in the telencephalon of toadsBufo bufo during stimulation with different visual key stimuli was quantitatively mapped by means of the¹⁴C-2DG autoradiographic method: (i) a 4×28 mm² worm-like stripe (W) eliciting prey catching responses, (ii) a 84×84 mm² square (S) releasing predator avoidance responses, and (iii) a 28×4 mm² antiwormlike stripe (A) eliciting no motor response.Various telencephalic structures changed¹⁴C- 2DG uptake statistical significantly during stimulation with the above visual objects in comparison with binocular enucleated animals (brain-to-brain comparison) and in comparison between both hemispheres in monocular animals (interhemispherical comparison): (1) The ventral two-thirds of the posterior half of the medial palliumdecreased ^14C-2DG uptake during W- and S-experiments, particularly in response to W. (2) In the posterior two-thirds of the lateral pallium,¹⁴C- 2DG uptake wasdecreased in response to the worm-, andincreased in response to the square (S) and antiworm stimuli (A). (3) The ventral striatumincreased uptake of¹⁴C-2DG during the animal's response to W- and S-stimuli significantly stronger than in the A-experiment. (4) The dorsal striatum also showed a significant change in¹⁴C-2DG uptake which, on a lower level, was not correlated with the type of stimulation experiment.Various prosencephalic structures are involved in circuitries related to attentional phenomena and the gating of prey catching and predator avoidance behavior. The different functions of these structures are discussed.Abbreviations A anterior dorsal thalamus - ACC nucleus ac-cumbens - APL amygdala, pars lateralis - APM amygdala, pars medialis - aLP anterior third of the lateral pallium - aMP anterior half of the medial pallium - B Bed nucleus of the palliai commissure - Ea entopeduncular nucleus, pars anterior - dMP dorsal medial pallium - dP dorsal pallium - dSTR dorsal striatum - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, postero-dorsal division - OT optic tectum - P posterior thalamic nucleus - pLP posterior two-thirds of the lateral pallium - PO preoptic area of the hypothalamus; - RET tegmental portion of the medial reticular formation - SEP medial (MS) and lateral (LS) septum - vMP ventral two-thirds of the medial pallium (MP) - vSTR ventral striatum     

Responses of medullary neurons to moving visual stimuli in the common toad

The concept of coded 'command releasing systems' proposes that visually specialized descending tectal (and pretectal) neurons converge on motor pattern generating medullary circuits and release--in goal-specific combination--specific action patterns. Extracellular recordings from medullary neurons of the medial reticular formation of the awake immobilized toad in response to moving visual stimuli revealed the following main results. (i) Properties of medullary neurons were distinguished by location, shape, and size of visual receptive fields (ranging from relatively small to wide), by trigger features of various moving configural stimulus objects (including prey- and predator-selective properties), by tactile sensitivity, and by firing pattern characteristics (sluggish, tonic, warming-up, and cyclic). (ii) Visual receptive fields of medullary neurons and their responses to moving configural objects suggest converging inputs of tectal (and pretectal) descending neurons. (iii) In contrast to tectal monocular 'small-field' neurons, the excitatory visual receptive fields of comparable medullary neurons were larger, ellipsoidally shaped, mostly oriented horizontally, and not topographically mapped in an obvious fashion. Furthermore, configural feature discrimination was sharper. (iv) The observation of multiple properties in most medullary neurons (partly showing combined visual and cutaneous sensitivities) suggests integration of various inputs by these cells, and this is in principle consistent with the concept of command releasing systems. (v) There is evidence for reciprocal tectal/medullary excitatory pathways suitable for premotor warming-up. (vi) Cyclic bursting of many neurons, spontaneously or as a post-stimulus sustaining event, points to a medullary premotor/motor property.