A trans-acting regulatory mutation that causes overproduction of phosphatidylserine synthase in Escherichia coli

We have isolated three mutants of Escherichia coli which have elevated levels of the phospholipid synthetic enzyme phosphatidylserine synthase. One of these strains carries a mutation, designated pssR1, which maps near minute 84 of the chromosome, distinct from the synthase structural gene (pss) at minute 56. The pssR1 mutation causes selective overproduction of phosphatidylserine synthase, since the levels of six other lipid synthetic enzymes are unaltered. The specific activity of the synthase in crude cell extracts of mutants harboring pssR1 is about five times greater than wild type. The synthase can also be overproduced 10-fold in wild type strains with hybrid ColE1 plasmids carrying the synthase structural gene (pss). A pssR1 mutant harboring such a pss plasmid overproduces the synthase about 50-fold. This multiplicative interaction of pssR1 and cloned pss demonstrates that pssR1 is trans-acting. The synthase has been purified in parallel from pssR1 and pssR+ strains. The pssR1 mutant yields more total synthase protein than pssR+, but the pure enzyme has the same specific activity in both cases. Therefore, pssR1 acts by increasing the amount of the normal protein, not by activating the enzyme. The discovery of pssR shows that there are regulatory loci which control the production of enzymes involved in membrane lipid synthesis.     

Configurations resulting from iso-chromatid and iso-subchromatid unions after meiotic and mitotic prophase irradiation

Summary By making use of the chromosomes of Trillium erectum as a model, potential and actual configurations arising from presumed iso-chromatid and iso-subchromatid unions after irradiation of meiotic or mitotic prophase have been studied and analyzed. Diagrams and photographs of various recognizable types of chromatid or subchromatid rearrangements are presented. A minimum of two iso-chromatid unions within an arm of a single chromosome in meiotic prophase, if separated by a single chiasma, can give rise to a monocentric chromosome with a triplicated segment, the middle portion of which is an inversion. A minimum of two iso-subchromatid breaks within an arm at either meiotic or mitotic prophase also can result in the production of a monocentric chromosome containing a triplicated segment. The stage of appearance of dicentrics or bridges arising from chromatid or subchromatid unions in meiotic prophase is influenced by chiasma number and pattern and by the number of strands per chromosome or chromatid. Some of the rearrangements described may have genetic and evolutionary implication of considerable potential importance which has not been recognized previously.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

SUB-CHROMATID REARRANGEMENTS IN TRILLIUM ERECTUM. I. ORIGIN AND NATURE OF CONFIGURATIONS INDUCED BY IONIZING RADIATION

Wilson , G. B. (Michigan State U., East Lansing.), A. H. Sparrow , and Virginia Pond . Subchromatid rearrangements in Trillium erectum. I. Origin and nature of configurations induced by ionizing radiation. Amer. Jour. Bot. 46(4): 309–316. Illus. 1959.—Microsporocytes of Trillium erectum were x-irradiated with 25 r at various stages of meiotic prophase and at first metaphase. Analysis of these cells at the following first and second anaphase revealed that post-pachytene irradiation produces 2-side-arm bridges which are indicative of half-chromatid exchanges. The occurrence of these bridges and knowledge of the structure and spatial relationship of chromatid strands in T. erectum have led to certain conclusions regarding the target and the number of strands broken by a single event: (1) the most likely target for primary effects is the 4 associated half-chromatids of a half-bivalent. The results of irradiation experiments suggest that the half-bivalent is effectively as well as structurally quadripartite at stages following pachytene. (2) Consideration of the configurations which would result from breakage and rejoining of 2, 3 or all 4 strands of the half-bivalent indicates that only 2 of the 4 half-chromatids are broken by a single event. Exchanges between 2 half-chromatids of sister chromatids will produce two recognizable types of 2-side-arm bridges: one with a true dicentric half-chromatid and one in which the bridge results merely from an interlocking of coils. Whether a 2-side-arm bridge appears at first or second meiotic anaphase is determined by the position and number of chiasmata between the point of breakage and the kinetochore. No 2-side-arm bridges have been detected at microspore anaphase following meiotic prophase irradiation. The types of configurations which might be expected at microspore metaphase as a result of broken 2-side-arm bridges are noted.     

Contraction of smooth muscle of pig airway tissues from before birth to maturity

Two heavy chains of smooth muscle myosin (MHC1 and MHC2) were identified in pig airways and parenchyma. The ratio of MHC1 to MHC2 was the same along the bronchial tree in animals of the same age, but it changed with age (mature, young, suckling, and fetus), ranging from 0.8 in the mature to 2.2 in the fetus. Stress developed in airway (trachea, bronchus, and bronchiole) and parenchymal preparations in response to carbachol and histamine (mN/mm2) was normalized for myosin content (N/mm2 myosin). Airways from sucklings always developed the greatest stress to carbachol and histamine with the rank order of maximum force (Emax) suckling greater than fetus greater than young greater than mature for carbachol in large airways. Airway ranking to histamine was similar except that Emax of fetal bronchus and bronchiole were least. In parenchymal strips, mature animals gave strong responses to carbachol and histamine compared with other age groups. Sensitivity to carbachol was increased in the suckling trachea; otherwise it did not vary with age. Chemically skinned tracheal fibers exhibited three- to fourfold greater sensitivity to Ca2+ in fetal and suckling airways compared with the older animals. It is concluded that maturation of smooth muscle occurs in the expression of myosin, in the Ca2(+)-force relationships of the contractile machinery, and in the pharmacological responsiveness of the intact smooth muscle, with the latter greatest at or soon after birth.     

The sight test fee: effect on ophthalmology referrals and rate of glaucoma detection.

OBJECTIVE--To assess changes, if any, in the numbers of referrals and outcome of glaucoma referrals to the hospital eye service since the introduction of the sight test fee on 1 April 1989. DESIGN--Review of referral records and clinical notes. SETTING--Referrals to the Bristol Eye Hospital. SUBJECTS--51,919 patients referred to the Bristol Eye Hospital between 1984 and 1992. 9438 case notes of patients referred between 1987 and 1991 were examined in detail. MAIN OUTCOME MEASURES--Numbers of referrals; rate of adult true positive glaucoma referrals. RESULTS--Referrals to the Bristol Eye Hospital were between 13.7% and 19.0% fewer than expected after the introduction of the sight test fee. True positive glaucoma referrals were reduced by the same proportion. CONCLUSIONS--The numbers of patients being identified as requiring treatment or follow up for potentially blinding glaucoma have declined by nearly one fifth since the introduction of the sight test fee. An increased prevalence of preventable blindness may result.     

Structural Comparisons of Muscle and Nonmuscle Actins Give Insights Into the Evolution of Their Functional Differences

Actin is a highly conserved protein although many isoforms exist. In vertebrates and insects the different actin isoforms can be grouped by their amino acid sequence and tissue-specific gene expression into muscle and nonmuscle actins, suggesting that the different actins may have a functional significance. We ask here whether atomic models for G- and F-actins may help to explain this functional diversity. Using a molecular graphics program we have mapped the few amino acids that differ between isoactins. A small number of residues specific for muscle actins are buried in internal positions and some present a remarkable organization. Within the molecule, the replacements observed between muscle and nonmuscle actins are often accompanied by compensatory changes. The others are dispersed on the protein surface, except for a cluster located at the N-terminus which protrudes outward. Only a few of these residues specific for muscle actins are present in known ligand binding sites except the N-terminus, which has a sequence specific for each isoactin and is directly implicated in the binding to myosin. When we simulated the replacements of side chains of residues specific for muscle actins to those specific for nonmuscle actins, the N-terminus appears to be less compact and more flexible in nonmuscle actins. This would represent the first conformational grounds for proposing that muscle and nonmuscle actins may be functionally distinguishable. The rest of the molecule is very similar or identical in all the actins, except for a possible higher internal flexibility in muscle actins. We propose that muscle actin genes have evolved from genes of nonmuscle actins by substitutions leading to some conformational changes in the protruding N-terminus and the internal dynamics of the main body of the protein. Received: 15 March 1996 / Accepted: 14 July 1996     

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XXVII. A FURTHER STUDY OF PHYSODERMA HYDROCOTYLIDIS VIÉGAS AND TEIXEIRA

A further study of Physoderma hydrocotylidis from California on its host Hydrocotyle ranunculoides gives further details on its structure and development. EM micrographs reveal that the notably thin wall of the resting spore consists of an outer, an intermediate, and an inner layer. The complete wall thickness from measurements of these EM preparations is only 0.5 μm, the thinnest of any known Physoderma resting spore. Such resting spores germinate readily in 2–5 h by the dehiscence of a broad cap and formation of a protruding endosporganium. Zoospores from the latter infect epidermal host cells and produce either the endobiotic, polycentric rhizoidal system with tenuous filaments, turbinate cells and eventually resting spores, or a hitherto unknown epibiotic, monocentric, rhizidiaceous sporangial stage. Zoospores from the latter may all bear colorless lipid globules or all faintly orange-colored ones.     

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.