Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage

Multiple endocrine neoplasis type 2A (MEN2A) is one of several kinds of cancers that appear to be inherited in an autosomally dominant fashion. We have assigned the MEN2A locus to chromosome 10 by linkage with a new DNA marker (D10S5). The linkage led us to investigate other chromosome 10 markers and demonstrate linkage between the disease locus and the interstitial retinol-binding protein (IRBP) gene. The D10S5 locus was sublocalized to 10q21.1 by hybridization in situ and the IRBP gene to p11.2----q11.2 with a secondary site at q24----q25. The linkages were established using 292 members of five families, three different restriction fragment length polymorphisms (RFLPs) at D10S5 and two RFLPs recognized by the IRBP probe. The recombination frequencies from pairwise linkage analysis between the disease and two marker loci D10S5 and IRBP were 0.19 and 0.11, with maximum lod scores of 3.6 and 8.0 respectively. Ordering of the three loci by multipoint analysis placed the IRBP gene approximately midway between the disease and D10S5 loci.     

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.     

Molecular genetic evidence for heterogeneity in manic depression

Manic depression is a severe cyclic mental illness that can be unipolar or bipolar and has a lifetime risk of approximately 7 per 1,000 in most populations. Families with multiple cases of manic depression have been described that are compatible with both autosomal dominant and X-linked modes of genetic transmission. Psychoactive antidepressant and stimulant drugs that help to ameliorate depression and mania are thought to act by affecting catecholamine neurotransmitter systems such as adrenaline, noradrenaline and dopamine, amongst others. Mutations affecting the tyrosine hydroxylase (TH) gene, which encodes the rate-limiting enzyme for the synthesis of these three neurotransmitters, might therefore be responsible for causing the manic depressive phenotype. We have studied three Icelandic kindreds amongst whom it appears that a single autosomal dominant disease allele is segregating. In these families there were 44 cases amongst 73 individuals at risk. Genetic linkage studies were carried out using clones encoding tyrosine hydroxylase the variable portion of the Harvey-ras-1 (HRAS1) locus and the variable region of the insulin gene (INS). All three markers are closely linked on chromosome 11 and were used to observe the segregation of restriction fragment length polymorphisms (RFLPs) in the three affected kindreds. We found no evidence for linkage to these markers in any of the three families. In contrast, Gerhard et al. found linkage between manic depression and HRAS1 in a single large Amish kindred. We conclude that there is genetic heterogeneity of linkage in manic depression. Therefore mutations at different loci are responsible for the manic depressive phenotype in the Amish and in Iceland.     

The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene

Amyloid beta-protein (AP) is a peptide of relative molecular mass (Mr) 42,000 found in the senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles of patients with Alzheimer's disease and Down's syndrome (trisomy 21). Recent molecular genetic evidence has indicated that AP is encoded as part of a larger protein by a gene on chromosome 21 (refs 5-7). The defect in the inherited autosomal dominant form of Alzheimer's disease, familial Alzheimer's disease (FAD), has been mapped to the same approximate region of chromosome 21 by genetic linkage to anonymous DNA markers, raising the possibility that this gene product, which could be important in the pathogenesis of Alzheimer's disease, is also the site of the inherited defect in FAD (ref. 5). We have determined the pattern of segregation of the AP gene in FAD pedigrees using restriction fragment length polymorphisms. The detection of several recombination events with FAD suggests that the AP gene is not the site of the inherited defect underlying this disorder.     

Myosin subfragment-1 is sufficient to move actin filaments in vitro

The rotating crossbridge model for muscle contraction proposes that force is produced by a change in angle of the crossbridge between the overlapping thick and thin filaments. Myosin, the major component of the thick filament, is comprised of two heavy chains and two pairs of light chains. Together they form two globular heads, which give rise to the crossbridge in muscle, and a coiled-coil rod, which forms the shaft of the thick filament. The isolated head fragment, subfragment-1 (S1), contains the ATPase and actin-binding activities of myosin (Fig. 1). Although S1 seems to have the requisite enzymatic activity, direct evidence that S1 is sufficient to drive actin movement has been lacking. It has long been recognized that in vitro movement assays are an important approach for identifying the elements in muscle responsible for force generation. Hynes et al. showed that beads coated with heavy meromyosin (HMM), a soluble proteolytic fragment of myosin consisting of a part of the rod and the two heads, can move on Nitella actin filaments. Using the myosin-coated surface assay of Kron and Spudich, Harada et al. showed that single-headed myosin filaments bound to glass support movement of actin at nearly the same speed as intact myosin filaments. These studies show that the terminal portion of the rod and the two-headed nature of myosin are not required for movement. To restrict the region responsible for movement further, we have modified the myosin-coated surface assay by replacing the glass surface with a nitrocellulose film. Here we report that myosin filaments, soluble myosin, HMM or S1, when bound to a nitrocellulose film, support actin sliding movement (Fig. 2). That S1 is sufficient to cause sliding movement of actin filaments in vitro gives strong support to models of contraction that place the site of active movement in muscle within the myosin head.     

Localization of the gene for familial adenomatous polyposis on chromosome 5

Colorectal cancer is the second most common cancer in the United Kingdom and other developed countries in the West. Although it is usually not familial, there is a rare dominantly inherited susceptibility to colon cancer, familial adenomatous polyposis (FAP; also often previously called familial polyposis coli). During adolescence affected individuals develop from a few hundred to over a thousand adenomatous polyps in their large bowel. These are sufficiently likely to give rise to adenocarcinomas to make prophylactic removal of the colon usual in diagnosed FAP individuals. Adenomas may occur elsewhere in the gastrointestinal tract and the condition is often associated with other extracolonic lesions, such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours. Adenomata have been suggested to be precancerous states for most colorectal tumours. Knudson has suggested that the mutation for a dominantly inherited cancer susceptibility may be the first step in a recessive change in the tumour cells, and that the same gene may be involved in both familial and non-familial cases of a given tumour. Following up a case report of an interstitial deletion of chromosome 5 in a mentally retarded individual with multiple developmental abnormalities and FAP, we have now shown that the FAP gene is on chromosome 5, most probably near bands 5q21-q22.     

Identification of receptor contact site involved in receptor-G protein coupling

The mammalian G proteins transduce information from extracellular signals, including neurotransmitters, hormones and sensory stimuli, into regulation of effector enzymes or ion channels within cells. Triggered by appropriate extracellular signals, receptor proteins specifically activate members of the G protein family by catalysing replacement of GDP by GTP at the guanine nucleotide binding site. Like the receptor proteins, the heterotrimeric G proteins exhibit impressive structural similarities, suggesting that all receptor-G protein interactions use homologous structural elements and a single molecular mechanism. Topologically equivalent portions of each G protein may therefore interact with the appropriate receptor. We recently predicted the secondary structure of a composite G protein alpha-chain and proposed that a predicted amphipathic alpha-helix at the extreme carboxy-terminus of the polypeptide directly contacts receptors. This proposal has now been confirmed by sequencing complementary DNAs of the gene that encodes the alpha-chain (alpha s) of the stimulatory regulator (Gs) of adenylyl cyclase in wild-type cells and in a mutant mouse S49 lymphoma cell line, unc, in which Gs cannot be activated by hormone receptors. The sequences reveal a point mutation in the unc gene that substitutes a proline residue for an arginine near the carboxy-terminus of the alpha s-polypeptide. Expression of recombinant alpha s-unc in genetically alpha s-deficient S49 cells reproduces the unc phenotype.     

A new immunomodulating compound (AS-101) with potential therapeutic application

There has been interest in the potential of synthetic compounds to modify immune responses by imitation of cytokine action. Direct administration of interleukin 2 (IL-2) in conjunction with adoptive transfer of lymphokine activated killer cells has been used in the treatment of cancer, but there are toxic effects resulting from the high doses of IL-2 required. We have developed a new synthetic compound, ammonium tri-chloro(dioxoethylene-O,O'-)tellurate (AS-101), which has immunomodulating properties and minimal toxicity. The effects of AS-101 on the activation and function of immunocompetent cells have been assessed. We have found that AS-101 induces proliferation and IL-2 production by human lymphocytes in vitro, and enhances the production of IL-2 and colony-stimulating factor by mouse spleen cells. Splenocytes of BALB/c mice injected with AS-101 increased production of IL-2 and CSF in vitro in the presence of mitogen. Mononuclear cells of normal donors acquired responsiveness to recombinant IL-2 and bound monoclonal antibody to IL-2 receptor after incubation with AS-101. Splenocytes of mice treated in vivo with AS-101 expressed high levels of IL-2 receptor. The stimulation of lymphocytes by AS-101 apparently involves an increase in intracellular free calcium. AS-101 administered systemically to mice mediated antitumour effects which could be attributable to its immunomodulatory properties. In addition, AS-101 could directly enhance the ratio of OKT4 to OKT8-positive cells in cultured mononuclear cells from AIDS (acquired immune deficiency syndrome) patients. These results indicate that AS-101 is potentially useful in the treatment of clinical conditions involving immunosuppression.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.