Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing

Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.     

Structural elements of bactopterin from Pseudomonas carboxydoflava carbon monoxide dehydrogenase

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.     

Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

Testing the “methanochondrion concept”: are nucleotides transported across internal membranes in Methanobacterium thermoautotrophicum?

In order to test the Methanochondrion concept, uptake of adenine nucleotides in various membrane preparations of Methanobacterium thermoautotrophicum was studied. The uptake showed properties which are in general interpreted as indicative of a transport mechanism: (i) kinetics in the time range of minutes, (ii) temperature dependence, (iii) substrate specificity and (iv) failure to remove the substrate by extensive washing.However, nucleotide transport as an interpretation of this uptake can definitely be excluded. Not only an exchange mechanism of the mitochondrial type, but also a general exchange or an uniport mechanism was ruled out. In contrast, the nucleotide uptake was shown to be actually a tight and specific binding of ADP and ATP to binding sites at the interior side of the cell membrane. This was conclusively demonstrated in protoplasts obtained from M. thermoautotrophicum cells. In these protoplasts which do not contain internal membranes also nucleotide binding was observed, but only after disruption of the plasma membrane by osmotic lysis, which leads to the exposure of binding sites.     

Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Spatial interactions in rapid pattern discrimination

We measured reaction times (RTs) for identification of a target among distracters under stabilized image conditions in which the positions of the target and the distracters were constant within a single experimental session. Under these conditions, the observer need not search for the target because its position is known. We nevertheless found that the presence of even a single distracter could elevate RTs. The magnitude of this effect depended on the distance of the distracter from the target and, for some observers, the distance of the distracter from the fovea. When we added not one but six background elements in a ring around the target, RT increased even more. If, apart from these neighboring distracters, the target was surrounded by more distracters located beyond the nearest neighbors, RT was, in general, not increased further. These findings suggest that adding background elements in a search task can elevate RTs in ways that are not dependent on the positional uncertainty of the target.     

The electrochemical proton potential generated by the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R₂S₄) or pentathionate (S₅O ₆ ⁼ ) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R₂S₄ reduction were 2,2-dithiobispropionate (R₂S₂) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R₂S₄. The electrical proportion () of the determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - R₂S_n (n=2–5) 2,2-polythiobispropionate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol - TPP tetraphenylphosphonium cation     

Recovery of rat mast cells after secretion: a morphometric study

Granule reconstitution in rat peritoneal mast cells following massive secretion was studied by morphometric techniques. Immediately following secretion, the earliest identifiable mast cells showed a substantial decrease in cell volume associated with granule loss. Cell volume then increased almost to the original level over a period of a month. The size of the Golgi apparatus increased markedly in the week following secretion and then returned to its original size. The total volume of granules increased slowly after the secretory depletion and by 34 days had not returned to the original value although the number of granules had recovered fully. The reconstitution of mast cells after secretion is a prolonged process with several phases resulting in mast cells of varying appearance and content. This heterogeneity generated by reconstitution post secretion must be considered in studies of populations of mast cells in vivo.