Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences

The specificity of monoclonal antibodies provides a powerful diagnostic and therapeutic tool in investigating human neoplasia. Radiological scanning and immunotherapy with mouse tumour-specific monoclonal antibodies have been applied to patients with some success, but a major problem is the neutralization of the mouse antibody induced by repeated administration of heterologous antibodies. To avoid or reduce such immune reactions, chimaeric immunoglobulins consisting of mouse variable (V) and human constant (C) regions can be synthesized. We have constructed a recombinant retrovirus DNA carrying genomic heavy-chain (H) variable-diversity joining (VH-D-JH) and C gamma 1 genes from different species and show here that the chimaeric intervening sequences are spliced out precisely. This procedure provides a useful method to construct the chimaeric mouse-human immunoglobulin gene to be expressed in Escherichia coli, yeast and animal cells. Unexpectedly, a hidden splice donor site in the 5'-flanking region of a human VH gene is used in place of the donor site of the leader sequence exon, resulting in the formation of the V region without the leader sequence.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

A new anatomy of the prestalk zone in Dictyostelium

The characteristic structure of the mature Dictyostelium culminant is created by the regionalized cellular differentiation and directed movement of prestalk cells. The front prestalk zone of the migratory slug has previously been considered to be a homogeneous tissue. Here we demonstrate, however, the existence of multiple classes of prestalk cells located in different parts or the slug anterior. The pDd56 and pDd63 genes encoding closely related extracellular matrix proteins are dependent for their expression upon DIF-1, the specific stalk-cell inducer. We have fused the promoters of the two genes to a modified chloramphenicol acetyltransferase (cat) gene to produce immunologically detectable proteins which localize to the cell nucleus. These two markers define three distinct kinds of 'prestalk' cells. One class, which we term 'prestalk A' cells, expressed the pDd63 gene. 'Prestalk B' cells express pDd56 and may also express the pDd63 gene. A third class, which we term 'prestalk 0' cells, expresses neither marker.     

Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs

Cell stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Of the DGK isoforms so far reported, only porcine DGK from lymphocytes has been characterized in detail. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C, and two E-F hand motifs, typical of Ca2(+)-binding proteins like calmodulin. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate.     

Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.     

Defining the resistance to oxygen transfer in tissue hypoxia

Studies of O2 supply in freshly isolated adult mammalian cells provide new insight into the factors that limit mitochondrial oxygenation in vivo. Of particular importance, mitochondria are present at high densities and often in apparent clusters, both of which contribute to local O2 gradients under hypoxic conditions. Current evidence indicates that the mitochondrial distribution is a component of the differentiated phenotype of adult mammalian cells and that specific motors and anchoring mechanisms are present to allow redistribution in response to developmental, physiological and pathological challenges. To compare the importance of resistance to O2 transfer under different conditions and at different sites along the supply path in vivo, a simple mathematical expression of relative resistance to O2 supply is introduced. Under various pathophysiological conditions, this resistance increases in specific regions of the pulmonary, circulatory or cellular supply path and results in O2 deficiency in the mitochondria. Regardless of cause, the relative resistance increases dramatically in the vicinity of mitochondrial clusters during hypoxia.     

Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although no central nervous system (CNS) abnormality has been identified. Recent studies have elucidated the molecular defect in DMD, including the absence of the protein dystrophin in affected individuals. Normal brain tissue contains dystrophin messenger RNA and dystrophin is present in low abundance in the brain and seems to be regulated in this tissue, at least in part, by a promoter that differs from that in muscle. Until now, antibodies and immunocytochemical methods used to demonstrate dystrophin at the plasma membrane of mouse and human muscle have proven inadequate to localize precisely dystrophin in the mammalian CNS. We have now made an antibody (anti 6-10) which is much more sensitive than those previously available to immunolabel dystrophin in the CNS. Using this antibody, we found that in the mouse, dystrophin is particularly abundant in the neurons of the cerebral and cerebellar cortices, and that it is localized at postsynaptic membrane specializations. Dystrophin may have a different role in neurons than in muscle, and an alteration at the synaptic level may be the basis of the cognitive impairment in DMD.     

Performance of an activated carbon made from waste palm shell in simultaneous adsorption of SO_x and NO_x of flue gas at low temperature

This study examined the individual and simultaneous adsorption of SOx(SO2)and NOx(NO-NO2)on activated carbon prepared from waste palm shell.The adsorption process was examined in a fixed bed reactor at low temperatures(100 300°C).For individual adsorption without any catalytic activation,SOx showed good adsorption whereas NOx was very much poor.In the simultaneous adsorption of SOx and NOx,SOx showed greater adsorption affinity than NOx.For palm shell activated carbon(PSAC)impregnated with metal catalyst(Ni and Ce)the concentration adsorbed profile showed that the amount of SOx adsorbed decreased regularly,while the amount of the adsorbed NOx increased irregularly.The properties of the pure and impregnated PSAC were analyzed by BET,SEM and EDX.These investigations indicated that PSAC impregnated with metal catalyst is the determining factor in the adsorption of SOx and NOx simultaneously.     

Electrical and band-gap properties of amorphous zinc-indium-tin oxide thin films

Zinc-indium-tin oxide (ZITO) films were grown by pulsed-laser deposition.Three different material compositions were investigated:ZITO-30,ZITO-50 and ZITO-70 in which 30％,50％ and 70％,respectively,of the...