Characterization of messenger RNA for aldolase B in adult and fetal human liver

High molecular weight cellular RNA was isolated from adult and fetal human liver tissue by a procedure of ethanol precipitation in concentrated guanidine-HCl solutions. About 5 mg of RNA were obtained from one gram of liver. RNA was fractionated by sucrose gradient ultracentrifugation. Aldolase B neosynthesized in a reticulocyte lysate cell-free system under the direction of total or fractionated RNA was purified by immunoaffinity microchromatography. Messenger RNA specifying synthesis of aldolase B exhibited a sedimentation coefficient of 16 S both in adult and fetal liver. This enzyme represented 1.3 % of the total neosynthesized proteins in adult liver, 0.1 % in the liver of a 6-month-old fetus and less than 0.01 % in the liver of a 4.5 month-old fetus.     

Molecules,fossils, and the origin of tetrapods

Summary Since the discovery of the coelacanth, Latimeria chalumnae, more than 50 years ago, paleontologists and comparative morphologists have debated whether coelacanths or lungfishes, two groups of lobe-finned fishes, are the closest living relatives of land vertebrates (Tetrapoda). Previously, Meyer and Wilson (1990) determined partial DNA sequences from two conservative mitochondrial genes and found support for a close relationship of lungfishes to tetrapods. We present additional DNA sequences from the 12S rRNA mitochondria gene for three species of the two lineages of lungfishes that were not represented in the first study: Protopterus annectens and Protopterus aethiopicus from Africa and Neoceratodus forsteri (kindly provided by B. Hedges and L. Maxson) from Australia. This extended data set tends to group the two lepidosirenid lungfish lineages (Lepidosiren and Protopterus) with Neoceratodus as their sister group. All lungfishes seem to be more closely related to tetrapods than the coelacanth is. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates. The common ancestor of lungfishes and tetrapods might have possessed multiple morphological traits that are shared by lungfishes and tetrapods [Meyer and Wilson (1990) listed 14 such traits]. Those traits that seem to link Latimeria and tetrapods are arguably due to convergent evolution or reversals and not to common descent. In this way, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. We recommended that the extinct groups of lobe-finned fishes be placed onto the molecular tree that has lungfishes and not the coelacanth more closely related to tetrapods. The placement of fossils would help to further interpret the sequence of morphological events and innovations associated with the origin of tetrapods but appears to be problematic because the quality of fossils is not always high enough, and differences among paleontologists in the interpretation of the fossils have stood in the way of a consensus opinion for the branching order among lobefinned fishes. Marshall and Schultze (1992) criticized the morphological analysis presented by Meyer and Wilson (1990) and suggest that 13 of the 14 morphological traits that support the sister group relationship of lungfishes and tetrapods are not shared derived characters. Here we present further alternative viewpoints to the ones of Marshall and Schultze (1992) from the paleontological literature. We argue that all available information (paleontological, neontological, and molecular data) and rigorous cladistic methodology should be used when relating fossils and extant taxa in a phylogenetic framework. Offprint requests to: Axel Meyer     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.     

Contrasts between subsurface microbial communities and their metabolic adaptation to polycyclic aromatic hydrocarbons at a forested and an urban coal-tar disposal site

The abundance and distribution of microorganisms and their potential for mineralizing polycyclic aromatic hydrocarbons (PAHs) were measured in subsurface sediment samples at two geographically separate buried coal-tar sites. At a relatively undisturbed forested site in the northeastern United States, metabolic adaptation to the PAHs was evident: Radiolabeled naphthalene and phenanthrene were converted to ¹⁴CO₂ in core material from inside but not outside a plume of groundwater contamination. However, at the urban site in the midwestern United States these PAHs were mineralized in sediments from both contaminated and uncontaminated boreholes. Thus, clear qualitative evidence showing an adaptational response by the subsurface microbial community was not obtained at the urban site. Instead, subtler clues suggesting metabolic adaptation by subsurface microorganisms from the urban site were discerned by comparing lag periods and extents of ¹⁴CO₂ production from radiolabeled PAHs added to samples from contaminated and uncontaminated boreholes. Despite slightly higher PAH mineralization activity in contaminated borehole samples, p-hydroxybenzoate was mineralized equally in all samples from the urban site regardless of location. No striking trends in the abundances of actinomycetes, fungi, and either viable or total bacteria were encountered. However, colonies of the soil bacterium, Bacillus mycoides, were detected on enumeration plates of several samples from unsaturated and saturated zones in both urban boreholes. Furthermore, other common soil bacteria, Myxococcus xanthus and Chromobacterium violaceum, were identified in samples from the uncontaminated urban borehole. The occurrence of bacteria usually restricted to surface soil, combined with the observation of fragments of building materials in many of the core samples, suggested that past excavation and backfilling operations may have caused mixing of surface soil with subsurface materials at the urban site. We speculate that this mixing, as well as non-coal-tar-derived sources of PAHs, contributed to the PAH-mineralizing activity present in the sediment samples from the uncontaminated urban borehole.     

Mental stress and cognitive performance do not increase overall level of cerebral O2 uptake in humans.

We measured cerebral metabolic rate of oxygen (CMRO2), cerebral blood flow (CBF), and cerebral lactate output during rest, during the execution of mental arithmetic, and during mental stress induced by physical and psychological annoyance. Measurements were performed in healthy volunteers by use of the Kety-Schmidt technique with 133Xe as the inert gas. Electroencephalographic desynchronization and highly significant increases in plasma catecholamines and heart rate verified that the test measurements were performed during conditions differing distinctly from the resting state. In accordance with an earlier study (Sokoloff et al. J. Clin. Invest. 34: 1101-1108, 1985), a minimal and nonsignificant 1% reduction of global CMRO2 during mental arithmetic was observed, signifying that this form of mental activation was unassociated with any detectable increase in overall cerebral synaptic activity. Mental stress induced a slight but highly significant (P less than 0.002) 6% reduction in global CMRO2. This finding is in contrast to results from earlier investigations and contradicts the generally accepted notion of an association between mental arousal and a diffuse upregulation of cerebral synaptic activity. During mental arithmetic and mental stress, cerebral lactate output increased by 207 and 344%, respectively, but because of large individual variations in the measured responses, the elevations reached statistical significance only during mental arithmetic.     

Protein HMG-17 is hyper-expressed in rat glucagonoma. Single-step isolation and sequencing

High-mobility-group protein 17 (HMG-17) was identified by reversed-phase high-performance liquid chromatography analysis as a major component in acidic extracts of transplantable rat glucagonoma tissue but not in insulinoma tissue of similar origin. The peptide was purified in a single step and the entire sequence of 89 amino acids was determined. Rat HMG-17 has a molecular mass of 9238 Da and shows strong similarity to human, bovine (94.4%) and chicken (88.8%) HMG-17. Six of the seven residues which vary among the mammalian sequences are located within a short segment (positions 64-83) present in the acidic, non-DNA-binding C-terminal part of HMG-17. This region shows least similarity to the otherwise related proteins HMG-14 and H6 (a trout HMG protein). Interestingly, four of the six variable positions are Asp in rat HMG-17 which results in an overall net increase in the negative charge of the C-terminal region. The nature of selective hyper-expression of HMG-17 in glucagon but not in insulin-producing tumor tissue remains to be clarified.     

Phagocytosis of erythrocytes by the proximal tubule of the rat kidney

Summary Morphological examination of kidney biopsies from patients with glomerulonephritis and hematuria has revealed the presence of erythrocytes within epithelial cells of the proximal tubule. This observation suggested that the proximal tubule might be capable of phagocytizing morphologically intact erythrocytes. To examine this possibility small quantities of heparinized autologous blood were injected into surface convolutions of proximal tubules of the rat kidney using standard micropuncture techniques. At time intervals ranging from 10 min to 120 h after injection, the kidneys were preserved for light and transmission electron microscopy by drip-fixation with a half-strength Karnovsky's glutaraldehyde-formaldehyde fixative.During the initial 6 h there was a flattening of the brush border and accumulation of electron-dense material representing hemoglobin in apical vacuoles and in lysosome-like structures. From 6 to 15 h after micropuncture, there was progressive loss of the brush border and the simultaneous formation of pseudopodia-like evaginations that extended from the apical plasma membrane and surrounded the individual erythrocytes. By 18 and 24 h, erythrocytes were observed in the proximal tubule cells. At later time intervals, edema, lymphocytic infiltration, and fibrosis were observed in the interstitium. In addition, crystalline structures were present in the lumen and the cells of both proximal and distal tubules. These findings suggest that in addition to their well-established ability to pinocytize hemoglobin and other proteins, the cells of the proximal tubule are capable of phagocytizing morphologically intact autologous erythrocytes. It is possible that phagocytosis by the proximal tubule cells may play a role in the disposal of erythrocytes from the tubular fluid in hematuric conditions.