The human apolipoprotein A-II gene is located on chromosome 1

Apolipoprotein (apo) A-II is a major constituent of high density lipoproteins (HDL). The gene for apoA-II has been localized to the p21----qter region of chromosome 1 in man by Southern blot hybridization analysis of DNA from human-mouse cell hybrids using a cloned human apoA-II cDNA probe. The regional assignment was established using two hybrids carrying a reciprocal translocation involving chromosomes 1 and 2. Comparison with previously established gene loci on chromosomes 1 suggests that apoA-II may reside in a conserved linkage group with renin and peptidase C. On the other hand, apoA-II is not linked to the apoA-I gene, which has been localized previously to chromosome 11.     

Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.     

Cytoplasmic effects on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of eight lines of hexaploid wheat (Triticum aestivum L. em. Thell) with different cytoplasms, the euplasmic nuclear donor Chinese Spring and seven alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. The calli were found to differ in their initial growth rates, their sensitivity to 2,4-D and their ability to organise shoot primordia, demonstrating that the cytoplasm can significantly affect the behaviour of tissues in culture. The potential for improving the responses of tissues in culture by cytoplasmic changes is noted.     

Localization of a beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to the long arm of human chromosome 17

We have assigned a human beta-crystallin gene, Hu beta A3/A1 (gene symbol: CRYB1), to chromosome 17 using a panel of 19 human-hamster somatic cell hybrids and blot-hybridization analysis of cell hybrid DNA. Positive probe-hybridization signal was detected in a hybrid that had lost the short arm of human chromosome 17 but retained the long arm, translocated to a hamster chromosome. In addition, in situ hybridization analysis of metaphase chromosome spreads of this cell line suggested that the most probable location for CRYB1 is on the long arm of chromosome 17, in the region q21.     

Studies of cell separation: A comparison of the osmotic response of human lymphocytes and granulocyte—Monocyte progenitor cells

The feasibility of using hypo- or hypertonic stress to selectively destroy lymphocytes while sparing stem cells was investigated. Lymphocytes were isolated from peripheral blood and exposed to Hanks' balanced salt solutions ranging in concentration from 66 to 2700 mOsm. The Boylevan't Hoff plot of cell volume versus reciprocal osmolality was linear. Following osmotic stress, viabilities of the lymphocytes and the granulocyte-monocyte progenitor cells (CFUc) were determined. Lymphocyte viability was assessed by tritiated thymidine incorporation following mitogen stimulation. CFUc viability was measured with the soft agar colony assay. Both types of cells were found to possess high osmotic tolerances compared to other blood cells. While progenitor cells in general appeared to survive anisotonic exposure somewhat better than lymphocytes, significant statistical differences were not established for most situations. The highest degree of CFUc enrichment was twofold, but there was a concomitant 50% drop in CFUc survival. These results suggest that osmotic stress is not a useful procedure for the separation of peripheral blood lymphocytes and stem cells.     

The mechanism of cobalamin binding to hog intrinsic factor

Chicken brain Arylsulfatase A (E.C.3.1.6.1) was immobilized by interaction with Concanavalin A. The immobilized enzyme retained its catalytic activity and this enzyme can be reused without appreciable loss of activity. The storage stability of bound and soluble enzymes was comparable and binding of enzyme to Concanavalin A increases its thermal stability. Kinetic studies indicated that bound enzyme shows similar anomalous kinetics as that of free enzyme but slight change was observed in relation to pH optima, Km value and activation energy.     

Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

Ca2+ transients induced by thyrotropin-releasing hormone rapidly lose their ability to cause release of prolactin

To investigate the relationship of changes in cytosolic free calcium concentrations [( Ca2+]c) caused by TRH to changes in PRL secretion, we simultaneously monitored PRL release and [Ca2+]c, using the fluorescent Ca2+ indicator indo-1, in freshly isolated perifused cells from rat anterior pituitary glands. We found that a 30-sec pulse of 100 nM TRH triggered a transient spike of [Ca2+]c, but prolonged PRL release for up to 30 min; continuous administration of TRH caused a sustained elevation in [Ca2+]c, but the same pattern and amount of PRL release as that caused by the pulse of TRH. PRL secretion was refractory to further pulses of TRH given at 10-min intervals for 40 min, but did respond to a second pulse of TRH given 40 min after the first pulse with no intervening pulses. Pulses of TRH given every 10 min still triggered spikes of [Ca2+]c of the same magnitude as the first pulse, indicating that the cause of the refractory state must occur at a post-receptor step that is after the mobilization of [Ca2+]c. A 30-sec pulse of a high concentration of KCl caused a transient spike of [Ca2+]c and transient, not prolonged, release. Additional pulses of KCl cause progressively less PRL release, although the magnitude of the spikes in [Ca2+]c did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cross-species comparison of the apolipoprotein B domain that binds to the LDL receptor

Apolipoprotein (apo)-B-100 is the ligand that mediates the clearance of low density lipoprotein (LDL) from the circulation by the apoB,E (LDL) receptor pathway. Clearance is mediated by the interaction of a domain enriched in basic amino acid residues on apoB-100 with clusters of acidic residues on the apoB,E (LDL) receptor. A model has been proposed for the LDL receptor binding domain of apoB-100 based on the primary amino acid sequence (Knott, T. J., et al. 1986. Nature. 323: 734-738). Two clusters of basic residues (A: 3147-3157 and B: 3359-3367) are apposed on the surface of the LDL particle by a disulfide bridge between Cys 3167 and 3297. Support for this single domain model has been obtained from the mapping of epitopes for anti-apoB monoclonal antibodies that block the binding of apoB to the LDL receptor. Here we test this model by comparing the nucleotide (from 9623 to 10,442) and amino acid sequence (from 3139 to 3411) of apoB-100 in seven species (human, pig, rabbit, rat, Syrian hamster, mouse, and chicken). Overall, this region is highly conserved. Cluster B maintains a strong net positive charge and is homologous across species in both primary and secondary structure. However, the net positive charge of region A is not conserved across these species, but the region remains strongly hydrophilic. The secondary structure of the region between clusters A and B is preserved, but the disulfide bond is unique to the human sequence. This study suggests that the basic region B is primarily involved in the binding of apoB-100 to the apoB,E (LDL) receptor.