In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

Early mouse embryos produce and release factors with transforming growth factor activity

Summary Previous studies have shown that extracts from mouse embryos at mid and late stages of development contain factors that exhibit transforming growth factor activity. The work reported here demonstrates that cultured mouse embryos at significantly earlier stages of development produce and release factors that exhibit the characteristic property of transforming growth factors. Specifically, the data demonstrate that embryos cultured from the blastocyst stage in serum-containing medium or in serum-free medium release factors that promote the anchorage-independent growth of normal rat kidney fibroblasts. It is shown that these factors are produced and released by cells derived from the inner cell mass and by trophoblasts. The precise developmental stage when production of these factors first begins has not been determined but our findings suggest that these factors are produced by cell types associated with early postimplatation embryos. This work was supported by the Laboratory of Viral Carcinogenesis at the National Cancer Institute and by grants from the National Cancer Institute (CA-36727) and the University of Nebraska Medical Center (22-271-732). Editor's Statement This paper presents evidence that, in an in vitro assay system, early embryonic cells are capable of both synthesizing and secreting TGF-like growth factors, implicating the production of these factors in the events of early development. David W. Barnes     

Zinc is a constituent of bovine heart cytochrome c oxidase preparations

Cytochrome c oxidase preparations from bovine heart muscle contain 1 zinc per 2 irons. Metal contents of nine preparations determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) show that Cu, Fe and Zn are the only metals present in significant amounts with average Cu/Fe, Fe/Zn, and Cu/Zn atom ratios of 1.3, 2.1 and 2.8, respectively. Removal of adventitious copper results in a Cu:Fe:Zn stoichiometry of 2:2:1. The zinc is tightly bound. Dialysis against a solution of 1,10-phenanthroline at pH 7.4 or an acidic buffer (pH 4.4) does not remove Zn. Dialysis against 0.8 M KCN at pH 10 causes partial loss of both Cu and Zn. This is the first evidence for the presence of Zn in a cytochrome c oxidase.     

In vitro metabolism of N-methyl-dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.     

Biomass production,total protein,chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes

Two green algae (Chlorella vulgaris and Scenedesmus obliquus) and four blue-green algae (Anacystis nidulans, Microcystis aeruginosa, Oscillatoria rubescens and Spirulina platensis) were grown in 81 batch cultures at different nitrogen levels. In all the algae increasing N levels led to an increase in the biomass (from 8 to 450 mg/l), in protein content (from 8 to 54 %) and in chlorophyll. At low N levels, the green algae contained a high percentage of total lipids (45 % of the biomass). More than 70 % of these were neutral lipids such as triacylglycerols (containing mainly 16:0 and 18:1 fatty acids) and trace amounts of hydrocarbons. At high N levels, the percentage of total lipids dropped to about 20 % of the dry weight. In the latter case the predominant lipids were polar lipids containing polyunsaturated C₁₆ and C₁₈ fatty acids. The blue-green algae, however, did not show any significant changes in their fatty acid and lipid compositions, when the nitrogen concentrations in the nutrient medium were varied. Thus the green but not the blue-green algae can be manipulated in mass cultures to yield a biomass with desired fatty acid and lipid compositions. The data may indicate a hitherto unrecognized distinction between prokaryotic and eukaryotic organisms.     

Characterization of Cholecystokinin from the Human Brain

Human forms of cholecystokinin have not previously been characterized chemically. In this study, we have extracted and purified the predominant molecular form of cholecystokinin present in human cerebral cortex. The peptide was characterized by amino acid analysis, automated peptide sequencing, and fast atom bombardment mass spectrometry. It appears to be identical to porcine cholecystokinin-octapeptide, with the sequence of Asp-Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe(NH2). This structural identity is consistent with the observations that the peptide in human brain and porcine cholecystokinin-octapeptide are recognized similarly by a battery of antisera to porcine cholecystokinin; that they coelute from several chromatographic systems, including gel filtration, ion exchange, and reversed-phase; and that they possess similar biological activities.     

Mass Spectrometric Analyses of Brain Synaptic Peptides Containing Taurine

The structure of a number of low-molecular-weight acidic peptides containing taurine prepared from calf brain synaptosomes and their subcellular vesicles was studied using electron impact mass spectrometry. At least seven sequences could be identified: N-acetylaspartyl-glutamyl-taurine, N-acetylaspartyl-taurine, N-acetylglutamyl-taurine, glutamyl-taurine, aspartyl-taurine, seryl-glutamyl-seryl-taurine, and seryl-taurine.     

Fatty acid composition and microbial activity of benthic marine sediment from McMurdo Sound, Antarctica

Abstract Signature lipids from the phospholipid esterlinked fatty acids (PELFA) of cell membranes were used to describe benthic microbial communities of 4 Antarctic sediments. Metabolic activities of the communities were determined by incorporation of [³H]thymidine into bacterial DNA and sodium [¹⁴C]acetate into membrane lipids. Biomass measurements from extractable phospholipid fatty acids per g dry wt. ranged between 6 to 76 nmol, or when converted to number of bacteria, 3.7 × 10⁸ to 4.5 × 10⁹ cells per g dry wt. The West Sound site at New Harbor contained the lowest biomass, while Cape Evans on the East Sound contained the greatest. A marked difference was also noted between sites in their sediment microbial community structure. The East Sound sites at Cape Armitage and Cape Evans contained a greater abundance of diatom marker lipids, whilst both sides of the Sound contained approximately the same relative amounts of bacterial groups distinguished using PELFA. Activity of sediment microorganisms measured by radiolabel incorporation under ambient conditions followed the trends of the biomass measurements. The East Sound sites were more active by an average of 45–73% for [³H]thymidine and possibly also for sodium [¹⁴C]acetate.     

Lack of prominent compensatory polycythemia in traditional native Andeans living at 4,200 meters

Red blood cell count (RBC), hemoglobin concentration ([Hb]) and hematocrit (Hct) were measured in 303 male Quechua children and adults, aged 6 to 57 years, living a lifestyle as traditional pastoralists and horticulturalists at a mean altitude of 4,200 m in the Southern Peruvian Andes. Values for RBC, [Hb], and Hct increased with age from middle childhood to young adulthood. However, among adults there was no significant association between age and any of these three parameters. Overall, there was approximately a 10-12% increase in the RBC, [Hb], and Hct above sea-level norms for all age groups. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) showed a slight but significant increase with age in children and adolescents, but the mean corpuscular hemoglobin concentration (MCHC) did not. We conclude that the study of highland Quechua Indians, living a traditional lifestyle as pastoralists and horticulturalists, does not support the long-held belief that altitude hypoxia provokes a dramatic compensatory polycythemia in healthy Andeans.     

Microbial fermentative preparation of L-[15N2]lysine and its tracer: application to serum amino acid kinetic studies

The microorganism Brevibacterium flavum 21129 has been used to produce multigram batches of L-[15N2]lysine of high purity and isotopic enrichment by supplementation of the growth medium with (15NH4)2SO4 of 98.0 atom% excess. The doubly 15N-labeled lysine can be detected at dilutions 10 times greater than singly labeled lysine when isotope dilution curves are analyzed by gas chromatography-mass spectrometry. This enhanced sensitivity permits kinetic measurements of plasma free-lysine isotope content over a 300-fold dilution during 6 h following a single oral bolus of 5 mg/kg body wt. This inexpensive preparation method lends itself to the production of highly useful biochemical compounds for kinetic studies of human nutrition.