Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II

Ribosomal protein S6 is a component of the eukaryotic 40S ribosomal subunit that becomes phosphorylated on multiple serine residues in response to a variety of mitogens, including insulin, growth factors, and transforming proteins of many oncogenic viruses. Recently, an activated S6 kinase (S6 K II) has been purified to homogeneity from Xenopus eggs, and characterized immunologically and at the molecular level. Purified S6 K II can be deactivated in vitro by incubation with either protein phosphatase 1 or protein phosphatase 2A. Reactivation and phosphorylation of S6 K II occurs in vitro with an insulin-stimulated microtubule-associated protein-2 (MAP-2) protein kinase which is itself a phosphoprotein that can be deactivated by protein phosphatase 2A. These studies suggest that a step in insulin signalling involves sequential activation by phosphorylation of at least two serine/threonine protein kinases.     

Multiple liquid crystal phases of DNA at high concentrations

DNA packaging in vivo is very tight, with volume concentrations approaching 70% w/v in sperm heads, virus capsids and bacterial nucleoids. The packaging mechanisms adopted may be related to the natural tendency of semi-rigid polymers to form liquid crystalline phases in concentrated solutions. We find that DNA forms at least three distinct liquid crystalline phases at concentrations comparable to those in vivo, with phase transitions occurring over relatively narrow ranges of DNA concentration. A weakly birefringent, dynamic, 'precholesteric' mesophase with microscopic textures intermediate between those of a nematic and a true cholesteric phase forms at the lowest concentrations required for phase separation. At slightly higher DNA concentrations, a second mesophase forms which is a strongly birefringent, well-ordered cholesteric phase with a concentration-dependent pitch varying from 2 to 10 micron. At the highest DNA concentrations, a phase forms which is two-dimensionally ordered and resembles smectic phases of thermotropic liquid crystals observed with small molecules.     

Self-tolerance eliminates T cells specific for Mls-modified products of the major histocompatibility complex

In mice the product of the Mlsa locus is an unusual antigen capable of interaction with certain products of the major histocompatibility locus (MHC) to form a ligand for a large portion of the T-cell alpha/beta receptor repertoire, including nearly all receptors that use V beta 8.1. The presence of Mlsa/MHC during T-cell development results in the deletion of T cells that express V beta 8.1, documenting the importance of clonal deletion in establishing tolerance to self antigens.     

Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome)

There have been many reports that eukaryotic cells contain ring-shaped 19S or 20S particles which are composed of numerous polypeptide subunits ranging in size between 25 and 35 kilodaltons. Because these particles seemed to copurify with inactive mRNA, they were assumed to function in regulating mRNA translation and hence were named 'prosomes' (for 'programmed-o-some'). A number of properties have been reported for these structures, including an association with specific RNA species or with certain heat-shock proteins and involvement in tRNA processing or aminoacyl tRNA synthesis. However, these proposed activities have not been supported by definitive evidence. During studies of the proteolytic systems in mammalian tissues, we noted many similarities between these 19S particles and the high molecular weight protease complexes that are present in most or all eukaryotic cells. This (700 kilodalton) enzyme complex, designated here as LAMP for 'large alkaline multi-functional protease', contains three distinct endoproteolytic sites which function at neutral or alkaline pH and are specific for hydrolysis of proteins, hydrophobic peptides, or basic peptides. This protease also exists in a latent form which can be activated by polylysine, fatty acids, or ATP. In this report, we show that the prosomes and these protease complexes are very similar or identical with respect to their size, polypeptide composition, immunological cross-reactivity, appearance in the electron microscope, radial symmetry of subunits, subcellular localization, and proteolytic activities. Therefore, the 'prosome' probably plays a critical role in intracellular protein breakdown, and we propose that it be renamed 'proteasome'.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins

Decay-accelerating factor (DAF), a glycoprotein that is anchored to the cell membrane by phosphatidylinositol, binds activated complement fragments C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes. Here, we report the molecular cloning of human DAF from HeLa cells. Analysis of DAF complementary DNAs revealed two classes of DAF messenger RNA, one apparently derived from the other by a splicing event that causes a coding frameshift near the C terminus. The apparent 'intron' sequence contains an Alu family member and encodes contiguous protein sequence. Two DAF proteins are therefore possible, having divergent C-terminal domains which differ in their hydrophobicity. Both mRNAs are found on polysomes, suggesting that both are translated. We propose that the major (90%) spliced DAF mRNA encodes membrane-bound DAF whereas the minor (10%) unspliced DAF mRNA may encode secreted DAF and we present expression data supporting this. The deduced DAF sequence contains four repeating units homologous to a consensus repeat found in a recently described family of complement proteins.     

An AIDS-related cytotoxic autoantibody reacts with a specific antigen on stimulated CD4+ T cells

Patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related conditions are known to have abnormalities of T cell subpopulations, including a decreased helper/inducer (bearing the CD4 antigen) to suppressor/cytotoxic (bearing the CD8 antigen) T cell ratio and decreased absolute numbers of T cells with the CD4+ phenotype. Infection of T cells with a retrovirus, termed human immunodeficiency virus (HIV), is thought to be important in these abnormalities. HIV infection alone does not adequately explain the CD4+ T-cell abnormalities seen in AIDS, however, and the nature of T-cell destruction in this disease remains poorly characterized. Here we describe an AIDS-related serum autoantibody that reacts with an antigen of relative molecular mass 18,000 (Mr 18K) restricted to lectin-stimulated or HIV-infected CD4+ T cells. The antibody also suppresses proliferation of CD4+ T cells in vitro and induces cytotoxicity of these cells in the presence of complement. Its role in the development of AIDS merits attention.     

No requirements of cyclic conformation of antagonists in binding to vasopressin receptors

Early reports that acyclic analogues of oxytocin and vasopressin (AVP) have drastically reduced agonistic activities established as dogma that an intact hexapeptide ring structure is essential for the pharmacological activities of analogues of neurohypophysial hormones. Thus, virtually all the many hundreds of agonistic and antagonistic analogues of the neurohypophysial peptides that have been reported contain an intact ring. Here we report that an intact ring is not essential for binding of antagonistic AVP analogues to vasopressor (V1) or antidiuretic (V2) AVP receptors. In fact, one acyclic AVP analogue seems to be about as potent as any previously reported cyclic V2 antagonist. This finding suggests new possibilities for the design of AVP analogues as pharmacological probes and for therapeutic use. Similar modifications might be useful in the design of analogues of other cyclic peptides, such as calcitonin, somatostatin and the atrial natriuretic factors.