Constitutive and inducible expression of a transgene directed by heterologous promoters in a trout liver cell line

Activities of heterologous promoters and enhancers in cultured rainbow trout liver cells were examined employing the bacterial chloramphenicol acetyltransferase gene as the reporter. SV40 promoter-enhancer and Rous sarcoma virus long terminal repeat directed constitutive expression at high levels. Moloney murine leukemia virus long terminal repeat and SV40 promoter combined with Adenovirus type 2 enhancer were also constitutively expressed. Drosophila Hsp70 promoter was activated when the transformed cells were cultured at 25 degrees C, a higher temperature than the temperature normally used, in faithful response to heat shock.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA.     

Fine structural studies on the reabsorption of colloid and fusion of colloid droplets in thyroid glands of TSH-treated mice

Summary The mechanism of the luminal colloid reabsorption and the fate of reabsorbed colloid droplets were studied ultracytochemically in epithelial cells of thyroid cells of TSH-treated mice. The luminal colloid is reabsorbed by micropinocytosis as well as phagocytosis into the follicle epithelial cell. Almost all the pinocytotic pits and vesicles are coated and often closely associated with actin filaments demonstrated by use of heavy meromyosin (HMM). This suggests the involvement of the actin filament system in making and transporting coated vesicles for micropinocytosis of the luminal colloid. Freeze-fracture images show aggregates of intramembrane particles on the P-face of the small depressions corresponding to the initial site for coated pits.The reabsorbed colloid droplets fuse with one another and with lysosomes. At the initial stage of this fusion, the limiting membranes of adjoining droplets fuse in a limited area to become pentalaminar, and then become trilaminar. Eventually, the membranes at the fusion point disappear, and the contents of both droplets become continuous. Freeze-fracture images reveal the disappearance of the intramembrane particles at the initial site where the fusion occurs.Examination of thin-sectioned tissue treated by rapid-freeze substitution fixation, shows clearly delineated cell organelles, and the rounded mitochondria have a characteristically high electron-dense matrix. Just beneath the limiting membrane of each colloid droplet, there always exists a low electron-dense layer about 10 nm thickness. The lysosomes are sometimes seen wrapped around the colloid droplet.This study was supported by grants (No. 56370002, No. 00544016) from the Japan Ministry of Education     

Simulation of adaptive modification of the vestibulo-ocular reflex with an adaptive filter model of the cerebellum

An adaptive linear filter model of the cerebellum (Fujita, 1982), which functions as a phase lead or lag compensator with learning capability, is applied to a problem of the cerebellar control of the vestibuloocular reflex (VOR). Under the assumption that the cerebellar flocculus accounts for adaptive modification of dynamic characteristics of the VOR, the cerebellar model was incorporated into a linear control model of the oculomotor system. The results of a simulation study are in good agreement with experimental data on eye movement.     

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wishar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI₂-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to Λ¹⁷-6-keto-PGF_1α, a stable metabolite of PGI₃, could not be detected by an incubation study of ¹⁴C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI₂-like substance produced by rat aorta is most likely to be PGI₂. itself and not PGI₃.     

Determination of the anti-ulcer agent geranylgeranylacetone in serum by gas chromatography—mass spectrometry

A highly specific and sensitive method for the determination of the anti-ulcer drug geranylgeranylacetone (GGA) in human serum is described. The extract from serum with hexane was saponified with potassium hydroxide and subjected to silica gel column chromatography to remove interfering substances. GGA in the partially purified extract was then reacted with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine and measured by selected ion monitoring using gas chromatography—mass spectrometry. A low detection limit (1 ng/ml) and high precision were obtained.     

Highly Sensitive Assay for Dopamine-β-Hydroxylase Activity in Human Cerebrospinal Fluid by High Performance Liquid Chromatography-Electrochemical Detection: Properties of the Enzyme

Abstract: This paper describes a new, sensitive assay for dopamine-β-hydroxylase (DBH) activity in human cerebrospinal fluid (CSF), serum and brain tissues by high performance liquid chromatography (HPLC) with electrochemical detection (ED). Dopamine (DA) was used as a substrate and was incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by a double-column procedure, the first column of Dowex-50-H⁺ and the second column of aluminum oxide. NE was adsorbed on the second aluminum oxide column and then eluted with 0.5 M-hydrochloric acid and assayed by HPLC-ED. Epinephrine (EN) was added to each incubation mixture as an internal standard, and this assay was therefore highly reproducible. The peak height in HPLC was linear from 500 fmol to 100 pmol of NE and EN. The lower limit of detection for NE formed enzymatically was about 30 pmol, which indicated that the sensitivity of this procedure was comparable to that of radioassay procedures. We applied the method to measurement of the activity of and examination of some of the characteristics of DBH in human CSF. DBH activity in CSF of Parkinsonian patients was lower than that of control patients. The properties of DBH in human CSF were similar to those in serum and adrenal medulla.     

Isolation of the diterpenoid teuflin (6-epiteucvin) from Teucrium viscidum var. miquelianum

The diterpenoid teuflin (6-epiteuevin) has been isolated from Teucrium viscidum var. miquelianum. Its base catalysed epimerization into teucvidin was studied under mild conditions and the pathway is discussed.     

An extra idic(15p)(q11) chromosome in Prader-Willi syndrome

Using a nonfluorescent AT-specific oligopeptide antibiotic, Distamycin A, on DAPI fluorescent banding of human chromosome (DA-DAPI) as described by Schweizer et al. (1978), we have detected an additional idic(15p) chromosome in a patient with typical Prader-Willi syndrome. On the basis of the evidence available in previous studies and of our own present results, we suspect that the fundamental genetic error in the syndrome is not caused by a chromosome aberration but by a gene aberration on chromosome 15.