Man's place in Hominoidea as inferred from molecular clocks of DNA

Summary Divergence dates among primates were estimated by molecular clock analysis of DNA sequence data. A molecular clock of -globin pseudogene was calibrated by setting the date of divergence between Catarrhini and Platyrrhini at 38 million years (Myr) ago. The clock gave dates of 25.3±2.4, 11.9±1.7, 5.9±1.2, and 4.9±1.2 Myr ago (± refers to standard error) for the separation of rhesus monkey, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. In placing confidence intervals of the estimates in a robust way, a bootstrap method was used. The 95% confidence intervals are 20.5–29.5, 9.0–14.8, 4.1–7.8, and 3.1–7.0 Myr ago for the separation of rhesus monkey, orangutan, gorilla, and chimpanzee, respectively. By a molecular clock dating of the Prosimii-Anthropoidea splitting, it was suggested that the evolutionary rate of the -globin gene was high early in primate evolution and subsequently decreased in the line of Anthropoidea. And, by a relative rate test using bootstrap sampling, the possibility of further decrease of the rate (more than 10%) in the line of Hominoidea compared with that of Cercopithecoidea was suggested. Therefore, the above dating of the splittings within Hominoidea may be biased slightly toward younger dates. On the other hand, mitochondrial DNA (mtDNA) seems to have evolved in mammals with a more uniform rate than the -globin gene. The ratio of the dates of orangutan splitting to chimpanzee splitting is larger for the mtDNA clock than that for the -globin clock, suggesting the possibilities of mt-DNA introgression among the early hominids and the early African apes, and/or of mtDNA polymorphism within the common ancestral species of orangutan and the African apes that obscures the date of the true species separation of orangutans.     

Mass Fragmentographic Determination of γ-Aminobutyric Acid and Glutamic Acid in Discrete Amygdaloid Nuclei of Rat Brain

A mass fragmentographic method for the simultaneous quantification of gamma-aminobutyric acid (GABA) and glutamic acid is described. In a convenient one-step reaction, the two amino acids were derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.     

Identification and Semi-Quantification of Gibberellins from the Pollen and Anthers of Zea mays by Immunoassay and GC/MS

Radioimmunoassays and enzyme-linked immunosorbent assays formethyl esters of gibberellins A₁, A₃, A₄, and A₇ were establishedusing an antiserum specific for GA₁-Me. The antiserum was characterizedby high titer and specificity for such C₁₉-GAs with 3ß-hydroxylgroup as GA₁, GA₃, GA₄ and GA₇. Combination of this antiserumand HPLC enabled us to identify and quantify GA, and GA₄ fromthe pollen of Zea mays with a high degree of reliability. Similarly,identification and quantification of GA₉ and GA₂₀ were alsomade possible by use of an antiserum specific for GA₂₀-Me. Combineduse of immunoassays and GC/MS enabled us to identify nine GAsfrom the pollen and four from the anthers of Zea mays. The identificationof non-13-hydroxylated GAs, such as GA₄ and GA₉, in additionto 13-hydroxylated GAs from the pollen and the anthers suggeststhat the early-non-hydroxylation pathway, as well as the early-13-hydrox-ylationpathway, operates in the male reproductive organs of Zea mays,and that the organ-specific biosynthesis and/or localizationof GAs in Zea mays is similar to that in Oryza saliva. (Received May 7, 1990; Accepted August 20, 1990)     

Enzymatic synthesis of γ-glutamyl-l-histidine by γ-glutamyltranspeptidase from Escherichia coli K-12

γ-Glutamyl- l -histidine was synthesized from l -glutamine and l -histidine by γ-glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12. The optimum conditions for the production of γ-glutamyl- l -histidine are determined. The yield of γ-glutamyl- l -histidine synthesized under the optimum conditions was 48% with both substrates (41.2 g/l). The product was isolated and then identified by an amino acid analyser, PMR spectrum and secondary ion mass spectrum analyses.     

Involvement of brain stem noradrenergic neurons in the development of hypertension in spontaneously hypertensive rats

This study attempted to investigate the possible involvement of the brain stem noradrenergic system in the development of hypertension in spontaneously hypertensive rats. Steady-state norepinephrine, dopamine, serotonin and 5-hydroxyindoleacetic acid concentrations and norepinephrine turnover were determined in the individual brain stem nuclei using high performance liquid chromatography with electrochemical detection. Decreased norepinephrine contents in the nucleus tractus solitarii in spontaneously hypertensive rats compared with Wistar-Kyoto rats at the age of 4, 8, and 16 weeks were demonstrated. In later stages (8 and 16 weeks), increased norepinephrine levels were observed in the nucleus reticularis gigantocellularis, the A1 and A5 areas. Norepinephrine turnover was not different between spontaneously hypertensive rats and Wistar-Kyoto rats in the nucleus tractus solitarii at the age of 4 and 16 weeks and increased in the nucleus reticularis gigantocellularis of spontaneously hypertensive rats at 16 weeks. Our results indicate that altered norepinephrine metabolism in the specific brain stem nuclei, especially the consistently decreased norepinephrine in the nucleus tractus solitarii of spontaneously hypertensive rats, contribute to the development of genetic hypertension.     

Decreased benzodiazepine receptor binding in epileptic El mice: A quantitative autoradiographic study

Benzodiazepine receptors and subtypes were examined in El mice and normal ddY mice with a quantitative autoradiographic technique. Specific [³H]flunitrazepam binding in stimulated El mice, which had experienced repeated convulsions, was significantly lower in the cortex and hippocampus than in ddY mice and unstimulated El mice. In the amygdala, specific [³H]flunitrazepam binding in stimulated El mice was lower than in ddY mice. There was a tendency for the [³H]flunitrazepam binding in these regions in unstimulated El mice to be intermediate between that in stimulated El mice and that in ddY mice, but there was no significant difference between unstimulated El mice and ddY mice. [³H]Flunitrazepam binding displaced by CL218,872 was significantly lower in the cortex of stimulated El mice than in that of the other two groups, and in the hippocampus of stimulated than of unstimulated El mice. These data suggest that the decrease in [³H]flunitrazepam binding in stimulated El mice may be due mainly to that of type 1 receptor and may be the result of repeated convulsions.     

Production of IGF-II-related peptide by an anaplastic cells line (AT-3) established from the dunning prostatic carcinoma of rats

Summary AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase in cell number; 71 ng at 3.0 × 10⁶ cells and 449 ng at 4.6 × 10⁷ cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture. These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media. Editor's statement One or more members of the insulin-like growth factor family have been established previously as mitogen for isolated prostate cells. This report suggests that IGF-II member of the family may be involved in autocrine support of cells from highly malignant prostate tumors.     

Formation of novel nitrofuran metabolites in xanthine oxidase-catalyzed reduction of methyl 5-nitro-2-furoate

After methyl 5-nitro-2-furoate was incubated with milk xanthine oxidase, three reduction products were isolated from the incubation mixture. Among them, two reduction products were new types of nitrofuran metabolites, i.e., metabolites 1 and 2 were identified as the dihydroxyhydrazine derivative (1,2-dihydroxy-1,2-di(5-methoxycarbonyl-2-furyl) hydrazine) and the hydroxylaminofuran derivative (methyl 5-hydroxylamino-2-furoate), respectively. Metabolite 3 was also identified as the aminofuran derivative (methyl 5-amino-2-furoate) by comparison with a synthetic sample.     

Intermittent hypoxia upregulates hepatic heme oxygenase-1 and ferritin-1, thereby limiting hepatic pathogenesis in rats fed a high-fat diet

Non-alcoholic fatty liver disease (NAFLD) is prevalent in patients with sleep apnea syndrome (SAS). Intermittent hypoxia (IH) and a high-fat diet (HFD) reproduce SAS and NAFLD, respectively, in rodents. In this study, rats were fed either an HFD or a standard diet (SD) for 2 weeks, and breathed either IH air or normoxic air for 4 days (early phase) or 6 weeks (late phase), with the same diets maintained during the exposure. HFD increased hepatic lipid accumulation, as detected by oil-red staining and triglyceride content. However, IH exposure reversed the hepatic steatosis at the late phase in these HFD-rats. IH exposure also increased hepatic expression of HO-1 and iron-binding protein ferritin-1 at the late phase, in association with increase in serum iron, bilirubin, and hepatic levels of lipid peroxides, such as 4-hydroxy-2-nonenal (HNE). IH exposure increased serum levels of hemoglobin (Hb) at the early phase and immunofluorescence of Hb and HO-1 in CD68-positive Kupffer cells (KCs) at the late phase. These findings support that IH induces erythrocytosis, erythro-phagocytosis, and generation of Hb in the KCs. The Hb promotes HO-1 expression in KCs, thereby produces iron, bilirubin, and carbon monoxide (CO). The iron would be either sequestrated by ferritin-1, transferred to the bone marrow for erythropoiesis, or would produce hydroxyradicals and HNE in the liver of rats fed an HFD. HNE might also contribute to the upregulation of HO-1, transferrin-1, and IκB, thereby limiting hepatic steatosis and inflammation via inhibition of nuclear factor κB (NFκB) activation.     

Reactive oxygen species stimulate mitochondrial allele segregation toward homoplasmy in human cells

Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)–induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H₂O₂), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation correlated with mtDNA-concatemer production and enabled transmission of multiple identical mt-genome copies as a single unit. Our results support a mechanism by which mt-allele segregation toward mt-homoplasmy is mediated by concatemers.