Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption-precipitation of [3H]leucine-labelled apo B. Over 50% (of 0.59 microgram/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein-associated, the remainder being membrane-bound. Lipoprotein-associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40%, with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.     

A new approach to chromosome doubling for haploid rice plants

Summary Rice nodal segments from three flowering haploids were excised and treated for different lengths of time with 0.3% or 0.4% colchicine (dissolved in 2% DMSO) in an attempt to induce fertile seeds. A combination of higher colchicine concentration and longer hours of treatment reduced the survival rate of treated segments, but more fertile plants were transformed. Pooled data showed that of the 842 segments used, 42.2% survived the treatment and sprouted, but only 31.9% were successfully established and grown to maturity. Among the 269 mature plants, 29,4% produced fertile seeds (panicles) with an average of 146.2 seeds per diploidized plant.     

Determination of desmosine, isodesmosine, and other amino acids by liquid chromatography with electrochemical detection following precolumn derivatization with naphthalenedialdehyde/cyanide

Naphthalenedialdehyde (NDA) in the presence of cyanide (CN) reacts with primary amines to produce fluorescent cyano[f]benzoisoindole (CBI) derivatives. These derivatives have been shown to be substantially more stable than the corresponding o-phthalaldehyde derivatives. However, one drawback of this method is that compounds derivatized at more than one site exhibit quenching, precluding the use of fluorescence detection. The CBI derivatives have been found to be electroactive and are oxidized at a modest oxidation potential (+750 mV). Electrochemical detection is especially useful for the analysis of compounds containing more than one primary amine site because the response is not attenuated as it is in fluorescence detection. Desmosine and isodesmosine were of particular interest because of their importance in elastic fiber and the lack of highly sensitive HPLC methods for the determination of these compounds. Both of these compounds react with NDA/CN to produce electrochemically active derivatives. The combination of derivatization with NDA/CN and electrochemical detection was found to be linear over three orders of magnitude. Detection limits for CBI-lysine and CBI-desmosine were 100 fmol at a S/N of 2. Amino acids in elastin were quantitated using this method. The results correlate well with what has been reported previously in the literature. A significant advantage of the use of liquid chromatography with electrochemical detection with precolumn derivatization with NDA/CN for the analysis of desmosine and isodesmosine is that they can be separated and quantitated individually using this method. In addition, the unique voltammetry of multiderivatized CBI-amino acids can be used to verify peak purity.     

Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.     

Termination region in rRNA genes from a eucaryotic thermophile, Thermomyces lanuginosus.

S1 mapping of the termination region in the ribosomal DNA from a thermophilic fungus, Thermomyces lanuginosus, revealed three distinct termini corresponding to the mature 25S rRNA, a precursor that is 19 nucleotides longer and corresponds to the 37S precursor in yeast cells, and a putative termination site at +96 that bears a limited sequence homology with the SalI box of mammalian cells. An estimate of the secondary structure suggested that the three termini are in close proximity, a feature that may be essential to precursor termination and maturation. The results raise questions regarding recently reported relationships between ribosomal DNA termination and spacer enhancer elements in fungi.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

Translational regulation of somatostatin in cultured sympathetic neurons

Coculture of sympathetic neurons with ganglion nonneuronal cells elevated levels of preprosomatostatin mRNA but did not alter neuronal synthesis, content, or release of somatostatin. Treatment of sympathetic neurons with culture medium conditioned by exposure to ganglion nonneuronal cells similarly elevated preprosomatostatin mRNA. Treatment with conditioned medium elevated somatostatin levels in pure neuronal cultures, but not in neurons cocultured with nonneuronal cells. Conditioned medium also failed to increase peptide levels in neurons cultured on a substratum of killed nonneuronal cells, despite a large increase in preprosomatostatin mRNA. These observations suggest that contact of sympathetic neurons with nonneuronal cell membranes inhibits the increase in peptide synthesis, but not the increase in preprosomatostatin mRNA after treatment with conditioned medium. Thus neuronal interactions with nonneuronal cells regulate somatostatin metabolism at both the mRNA and peptide levels. Regulatory effects on the mRNA and the peptide are separable and do not necessarily occur in parallel, and translational controls may be the rate-limiting factors.