Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH₂-terminal β-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively.     

Characterization and quantitation of fatty acids covalently bound to erythrocyte membrane proteins: anion transporter contains 1 mol of fatty acid thiol ester

Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein.     

The Engelbreth-Holm-Swarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans

Glycosaminoglycans were prepared from the Engelbreth-Holm-Swarm mouse tumor. Enzymatic analysis demonstrated heparan sulfate (95.8%) and chondroitinase ABC-sensitive galactosaminoglycans (4.2%). HPLC analysis of the disaccharide units showed that heparan sulfate chains were undersulfated on average, comprising approximately 30% nonsulfated and 60% N-sulfated disaccharide units with small proportions of other monosulfated and disulfated disaccharide units. In contrast, galactosaminoglycan chains were oversulfated, containing an appreciable proportion (15%) of a 4,6-disulfated (so-called E-type) disaccharide unit in addition to 51% of a 4-sulfated, 22% of a 6-sulfated, and 11% of a nonsulfated disaccharide unit. The significance of the oversulfated disaccharide structure is discussed in relation to the possible regulation of functions of hybrid proteoglycans from which the galactosaminoglycan chains are derived.     

Interleukin-1 inhibits the secretion of gastric acid in rats: possible involvement of prostaglandin

To examine the hypothesis that interleukin-1 may inhibit the secretion of gastric acid, the present study was carried out using pylorusligated rats. Based upon three lines of evidence, we report here that interleukin-1, both endogenously released and exogenously administered, suppresses gastric acid secretion and that the interleukin-1-induced inhibition of acid output is possibly mediated by prostaglandin. First, lipopolysaccharide, a potent stimulant of the release and production of endogenous interleukin-1, caused the suppression of gastric acid, and this response was dose-related. Second, the intraperitoneal injection of interleukin-1 resulted in a dose-related inhibition of gastric acid output. Third, the administration of indomethacin completely blocked the suppression of gastric acid secretion induced by interleukin-1. These results demonstrated for the first time that IL-1 might be involved in the regulation of gastric secretion.     

Cytogenetic analysis of 400 sperm from three translocation heterozygotes

Summary Sperm chromosome complements were studied in three men who carried reciprocal translocations. A total of 400 sperm were karyotyped after in vitro penetration of hamster eggs: 217 sperm from t(2;9) (q21;p22), 164 from t(4;6) (q28;p23) and 19 from t(7;14) (q21;q13). All possible 22 and 31 meiotic segregations were observed for t(2;9) and t(4;6); for t(7;14) only 22 segregations were observed. For alternate segregations, the number of normal sperm was not significantly different from the number of sperm carrying a balanced form of the translocation in any of the translocations, as theoretically expected. The percentage of sperm with an unbalanced form of the translocation was 57% for t(2;9), 54% for t(4;6) and 47% for t(7;14). There was no evidence for an interchromosomal effect in any of the translocations since the frequencies of numerical abnormalities (unrelated to the translocation) were within the normal range of control donors. The frequencies of X- and Y-bearing sperm did not differ significantly from 50%. Results from a total of 17 reciprocal translocations studied by sperm chromosomal analysis were reviewed.     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Recent problems in cell culture systems using normal human cells

Some remarkable studies of human cell culture have contributed to many basic and applied sciences on "normal human cells"; developments of biological products or physiologically activated substances. In this reviews, some problems concerning in vitro culture systems were discussed and recent advances on the researches of normal human cells were described.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

cDNA cloning of a novel heterogeneous nuclear ribonucleoprotein gene homologue in Caenorhabditis elegans using hamster prion protein cDNA as a hybridization probe.

The mammalian prion protein (PrPc) is a cellular protein of unknown function, an altered isoform of which (PrPsc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. The evolutionary conservation of the PrP gene has been reported in the genomes of many vertebrates as well as certain invertebrates. In the genome of nematode Caenorhabditis elegans, the sequence capable of hybridizing with the mammalian PrP cDNA probe has been demonstrated, predicting the presence of the PrP gene homologue in C.elegans. In this study, Southern analysis with the hamster PrP cDNA (HaPrP) probe confirmed the previous observation. Moreover, Northern analysis revealed that the sequence is actively transcribed in adult worms. Thus, we screened C.elegans cDNA libraries with the HaPrP probe and isolated a cDNA that hybridizes to the same sequence in C.elegans that hybridized with the HaPrP probe in the Southern and Northern analyses. The deduced amino acid sequence of this cDNA, however, is substantially homologous with heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins rather than mammalian PrPc. The hnRNPs contain the glycine-rich domain in the C-terminal half of the molecule, which also seemed to be in PrPc at the N-terminal half of the molecule. Both of the glycine-rich domains are composed of tracts with high G + C content, indicating that these tracts may due to the hybridizing signals. These results suggest that this cDNA clone is derived from a novel hnRNP gene homologue in C.elegans but not from a predicted PrP gene homologue.     

Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction

Summary Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the-- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.