Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells

Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.     

Identification of a new class of steroid hormone receptors

The gonads and adrenal glands produce steroids classified into five major groups which include the oestrogens, progestins, androgens, glucocorticoids and mineralocorticoids. Gonadal steroids control the differentiation and growth of the reproductive system, induce and maintain sexual characteristics and modulate reproductive behaviour. Adrenal steroids also influence differentiation as well as being metabolic regulators. The effects of each steroid depend primarily on its specific receptors, the nature of which could therefore provide a basis for classification of steroid hormone action. The successful cloning, sequencing and expression of the human glucocorticoid (hGR) (ref. 1), oestrogen (hER), progesterone (hPR), and mineralocorticoid (hMR) receptors, complementary DNA, plus homologues from various species, provides the first opportunity to study receptor structure and its influence on gene expression. Sequence comparison and mutational analysis show structural features common to all groups of steroid hormone receptors. The receptors share a highly conserved cysteine-rich region which functions as the DNA-binding domain. This common segment allows the genome to be scanned for related gene products: hMR cDNA for example, was isolated using an hGR hybridization probe. In this study, using the DNA-binding domain of the human oestrogen receptor cDNA as a hybridization probe, we have isolated two cDNA clones encoding polypeptides with structural features suggestive of cryptic steroid hormone receptors which could participate in a new hormone response system.     

Noradrenaline contracts arteries by activating voltage-dependent calcium channels

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.     

In vitro development ofXenopus skin glands producing 5-hydroxytryptamine and caerulein

The granular glands of amphibian skin synthesize and store a large amount of bioactive amines and peptides which are structurally similar to mammalian brain-gut peptides. To investigate the development of peptide- and amine-producing cells in the granular glands, pieces of dorsal skin taken at various stages fromXenopus laevis tadpoles were cultured, and the contents of caerulein and 5-hydroxytryptamine (5-HT) were measured. When pieces of skin from tadpoles at stages 57 to 60 (Nieuwkoop and Faber stages) were cultured in a medium containing 10% fetal calf serum (FCS medium) or one containing FCS treated with charcoal (chFCS medium), the caerulein and 5-HT levels were increased for the six days of the incubation period. The caerulein content was lower in the chFCS medium than in the FCS medium. Addition of thyroxine to the chFCS medium had no significant effect on the caerulein content. These results show that the caerulein-and 5-HT-producing cells of the granular glands can develop in a culture system with FCS- or chFCS-containing media, and suggest that FCS contains substances which are absorbed by charcoal and stimulate development of the amine- and peptideproducing cells of the glands. In a preliminary search for correlation between caerulein and 5-HT synthesis, addition of 5-hydroxytryptophan (5-HTP), a precursor to 5-HT, to the FCS medium increased 5-HT content and, conversely, caused significant decrease in caerulein content, suggesting that accumulation of caerulein in the granular glands is influenced by the amount of 5-HT synthesis. These studies indicate that this culture system is a useful model for investigating the development of peptide- and amine-producing cells.     

C_(301)铜基催化剂上CO、CO_2和H_2合成甲醇反应模式的研究

目前,由CO、CO_2和H_3合成甲醇反应的模式尚未能得出一致的观点。分歧主要在于合成过程中CO_2的作用。至七十年代中期,看法较为一致的模式为近年来,苏联学者采用动力学实验法和同位索示踪原子法对苏产CHM-1铜基催化剂进行研究,提出了另一种模式因而,为阐明甲醇合成反应模式,就必须阐明究竟甲醇是由CO、还是由CO_2 直接生成的。这不仅对于甲醇合成过程的优化,而且也对含CO_2工业废气的综合利用都是有意义的。本文在对国产C_(301)(Cu,ZnO和Al_2O_3组成)铜基催化剂进行动力学考察的基础上,研究CO_2在合成甲醇反应过程中的作用。实验压力5MP3,温度218—260℃,接触时间0.16—3.5秒,分别对仅含CO_2或CO和含CO、CO_2三种起始反应混合物气体进行了合成甲醇反应动力学行为的实验测定。结果表明:反应是一复杂的过程。对于含CO_2、H_2、Ar的混合物,由CO_2可以直接合成甲醇, 对含CO、H_2和Ar的混合物,甲醇由CO直接合成;而对既含CO_2又含CO 的混合物,甲醇同时由CO和CO_2合成。     

T gamma protein is expressed on murine fetal thymocytes as a disulphide-linked heterodimer

During the search for genes coding for the mouse alpha and beta subunits of the antigen-specific receptor of mouse T cells we encountered a third gene, subsequently designated gamma. This gene has many properties in common with the alpha and beta genes, somatic assembly from gene segments that resemble the gene segments for immunoglobulin variable (V), joining (J) and constant (C) regions; rearrangement and expression in T cells and not in B cells; low but distinct sequence homology to immunoglobulin V, J and C regions; other sequences that are reminiscent of the transmembrane and intracytoplasmic regions of integral membrane proteins; and a cysteine residue at the position expected for a disulphide bond linking two subunits of a dimeric membrane protein. Despite these similarities the gamma gene also shows some interesting unique features. These include a relatively limited repertoire of the germ-line gene segments, more pronounced expression at the RNA level in immature T cells such as fetal thymocytes and an apparent absence of in-frame RNA in some functional, alpha beta heterodimer-bearing T cells or cultured T clones and hybridomas. To understand the function of the putative gamma protein it is essential to define the cell population that expresses this protein. To this end we produced a fusion protein composed of Escherichia coli beta-galactosidase and the gamma-chain (hereafter referred to a beta-gal-gamma) using the phage expression vector lambda gt11 and raised rabbit antisera against the gamma determinants. Using the purified anti-gamma antibody we detected a polypeptide chain of relative molecular mass 35,000 (Mr 35K) on the surface of 16-day old fetal thymocytes. The gamma-chain is linked by a disulphide bridge to another component of 45K. No such heterodimer was detected on the surface of a cytotoxic T lymphocyte (CTL) clone 2C from which an in-phase gamma cDNA clone was originally isolated.     

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.     

Millisecond X-ray diffraction and the first electron density map from Laue photographs of a protein crystal

Attempts to use X-ray crystallography to extract three-dimensional information on transient phenomena in crystals have been hampered primarily by long data collection times. Here we report on the first difference Fourier map obtained from Laue diffraction photographs of a protein crystal, glycogen phosphorylase b. Data collection time was 3 s using the high-intensity white X-radiation generated on the wiggler magnet of the Daresbury Synchrotron Radiation Source (SRS), but data acquisition in the millisecond-submillisecond range is possible. The method presented here uses a simple difference technique and was designed to analyse structural changes relative to a known starting structure. The combination of this approach with cine techniques allows the recording of three-dimensional motion pictures at atomic resolution and opens up new areas in structural biology and chemistry.     

The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245

Chronic granulomatous disease (CGD) is a rare inherited disorder associated with a profound predisposition to infection due to the lack of a microbicidal oxidase system in the phagocytes of these patients. This syndrome is most commonly inherited through a defect on the X chromosome and the only clearly defined component of the oxidase system, the very unusual cytochrome b (b-245), has been shown to be missing from the cells of these patients. This cytochrome is a heterodimer composed of an alpha-chain of relative molecular mass (Mr) 23,000 (23K) and a 76-92K beta-chain; neither are detectable in neutrophils from X-linked CGD subjects. The defective X-CGD gene has recently been cloned by 'reverse genetics' but the protein predicted from the proposed complementary DNA sequence was not identified. We have purified the beta-chain of the cytochrome and sequenced 43 amino acids from the N terminus. Almost complete homology was obtained between this sequence and that of the complementary nucleotides 19-147 of the sequence of the X-CGD gene, originally designated as a non-coding region.