In situ demonstration of the activity of 3 beta-hydroxysteroid dehydrogenase on steroid hormone secreting cells within the paraaortic lymph nodes of golden hamster

The in situ activity of 3 beta-hydroxysteroid dehydrogenase was detected in clustered cells which showed steroid-producing morphology, within the capsule of the paraaortic lymph node. In light and electron microscopic studies, the positive reaction products were detected on intracapsular cell clusters. This result indicates that these unique cells may have a steroid secreting function within the lymph nodes.     

Coexistence of A- and B-form DNA in a single crystal lattice

It is well known that DNA can exist in a variety of conformations which can be interconverted by relatively mild changes in conditions. The in vivo conformation of DNA is usually thought to be the B form, but there is recent evidence that other conformations may be important in DNA-protein recognition. Different fragments of DNA crystallized under virtually identical conditions can form A, B or Z helices. A fragment that adopted an A conformation in a crystal was found in the B conformation in solution, whereas NMR spectroscopy of A-DNA films revealed the presence of a substantial amount of disordered B-DNA. Until now, however, a DNA fragment of a given sequence has not been crystallized in more than one global conformation. We report here an X-ray diffraction study of crystals of the DNA octamer dGGBrUABrUACC. In addition to a 'framework' of A-DNA, which gives discrete X-ray reflections, there are partially disordered B-DNA helices, recognized by their diffuse scattering features.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

Sequence homology of the LFA-1 and Mac-1 leukocyte adhesion glycoproteins and unexpected relation to leukocyte interferon

Cell-surface adherence reactions are fundamental to the biology of lymphocytes, monocytes and granulocytes. The lymphocyte function-associated 1 (LFA-1) and macrophage 1 (Mac-1) glycoproteins mediate differing types of adhesion reactions on these cells. LFA-1 participates in T-lymphocyte and natural killer-cell adhesion to target cells, whereas the Mac-1 antigen is identical to the complement receptor type 3, which mediates adhesion of monocytes and granulocytes to C3bi-sensitized particles. Deficiency of these proteins, in a heritable disease, results in multiple adhesion-related leukocyte defects. LFA-1 and Mac-1 resemble one another in overall structure, having alpha-subunits of relative molecular mass (Mr) 180,000 and 170,000, respectively, which are non-covalently associated with beta-subunits of Mr 95,000 in alpha 1 beta 1 complexes. Peptide mapping and immunological cross-reactivity have shown that the beta-subunits are highly related if not identical, but have revealed no similarities between the alpha-subunits. Nonetheless, the shared beta-subunit suggested that LFA-1 and Mac-1 might be members of a protein family containing diversified but evolutionarily related alpha-subunits. Therefore, we examine here the structure of the alpha-subunits by N-terminal amino-acid sequencing. Sequence homology shows that the alpha-subunits are members of a novel leukocyte adhesion protein family, and suggests that their evolution occurred by gene duplication. A search for similarities to previously sequenced proteins reveals a further unexpected homology between LFA-1 and leukocyte (alpha) interferons.     

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.