Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Morphological and immunocytochemical features of the pineal organ of C3H and C57BL mice at different stages of postnatal development

Considerable progress is currently being made in elucidating the molecular basis of the circadian (photoneuroendocrine) system by use of transgenic mice generated from the inbred strains C57BL and C3H. As in all other vertebrate species, the pineal organ is an important component of the photoneuroendocrine system in these mouse strains, but very little is known about its morphological and immunocytochemical features. We therefore investigated the pineal organ and the adjacent epithalamic region of adult, 10-, and 5-day-old C57BL and C3H mice for S-antigen, serotonin, and dopamine-ß-hydroxylase (DBH) immunoreactions. In adult animals, the pineal organ was more than 2 times bigger in C3H than in C57BL mice. In younger animals, this difference was already evident, but less pronounced. The S-antigen immunoreactivity was more intense in adult C3H than in C57BL mice. This difference developed with increasing age; it was not yet detectable in 5-day-old animals. The intensity of the serotonin immunoreaction was similar in both strains at all stages investigated. However, the serotonin immunoreaction was more pronounced in adult than in young animals. The relative DBH-immunoreactive area (used as a marker for the sympathetic innervation of the pineal organ) was much bigger in C3H than in C57BL mice; within each strain it remained relatively constant during postnatal development. Adult individuals of both strains contained S-antigen- and serotonin-immunoreactive cells in the habenular complex. Their number increased with age, but they were always more numerous in C3H. In conclusion, the study has shown considerable differences in pineal morphology between C3H and C57BL, which may be related to the well-known differen- ces in melatonin formation between these two strains.     

Tetramethylammonium:coenzyme M methyltransferase system from Methanococcoides sp.

A methanogen (strain NaT1) that belongs to the family of Methanosarcinaceae and that can grow on tetramethylammonium as the sole energy source has recently been isolated. We report here that cell extracts of the archaeon catalyze the formation of methyl-coenzyme M from coenzyme M and tetramethylammonium. The activity was dependent on the presence of Ti(III) citrate and ATP, and was rapidly lost under oxic conditions. Anoxic chromatography on DEAE-Sepharose revealed that two fractions, fractions 3 and 4, were required for activity. A 50-kDa protein that together with fraction 3 catalyzed methyl-coenzyme M formation from tetramethylammonium and coenzyme M was purified from fraction 4. From fraction 3, a 22-kDa corrinoid protein and a 40-kDa protein exhibiting methylcobalamin:coenzyme M methyltransferase (MT2) activity were purified. The N-terminal amino acid sequences of these purified proteins were determined. The 40-kDa protein showed sequence similarity to MT2 isoenzymes from Methanosarcina barkeri. Cell extract of strain NaT1 grown on trimethylamine rather than on tetramethylammonium did not exhibit tetramethylammonium:coenzyme M methyltransferase activity. The strain was identified as belonging to the genus of Methanococcoides, its closest relative being Methanococcoides methylutens. Received: 7 April 1998 / Accepted: 26 June 1998     

Juvenile open angle glaucoma: fine mapping of the TIGR gene to 1q24.3–q25.2 and mutation analysis

Autosomal dominant juvenile open angle glaucoma (JOAG) is an early-onset form of primary open angle glaucoma (POAG), which has been linked to chromosome 1q21–q31. Recently, mutations in the trabecular meshwork inducible glucocorticoid response gene (TIGR), one of the candidate genes mapped in this region, were identified in glaucoma patients of several families. We screened for mutations of the TIGR gene in two German families with JOAG and in 100 unselected sporadic cases of POAG. In the first family we identified a Pro370Leu mutation and in the second family a Gly367Arg mutation cosegregating with the glaucoma phenotype. No pathogenic mutation was found in 100 sporadic cases but a Tyr347Tyr polymorphism was found in two patients. Furthermore, fluorescence in situ hybridization (FISH) analysis was used to map a TIGR-specific yeast artificial chromosome to 1q24.3–q25.2. Received: 19 June 1997 / Accepted: 12 August 1997     

Physiological and Molecular Biological Characterization of Ammonia Oxidation of the Heterotrophic Nitrifier Pseudomonas putida

The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate. Product formation was accompanied by a small but significant release of NO, whereas N₂O evolution could not be detected under the assay conditions employed. The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions. Aerobically grown cells utilized γ-aminobutyrate as a carbon source and as a N-source by ammonification. The physiological experiments, in particular the inhibition pattern by C₂H₂, indicated that P. putida expressed an ammonia monooxigenase. DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Received: 9 April 1998 / Accepted: 15 May 1998     

Studies on the reaction of fluorescamine with primary amines

Fluorescamine is a useful reagent for the fluorometric assay of primary amines. The extent of the reaction between fluorescamine and primary amines, as well as the fluorescence intensities of the resulting fluorophors depend on pH, solvent composition and reagent concentration. Optimum values for these variables further depend on the amine under study. The influence of these parameters on the fluorogenic reaction of representative amines, and on their fluorophoric derivatives has been investigated, and the results are reported here.     

Cryptogenic Cerebral Embolism in Women Taking Oral Contraceptives

Fourteen women taking oral contraceptives were admitted during a five-year period because of acute cerebrovascular lesions. A diagnosis of major cerebral embolism was established in four of them. No source of embolism was found, and thorough investigation failed to reveal any predisposing illness. Cerebral embolism was a probable diagnosis in several of the remaining 10 patients. A comparison was made with the strokes occurring in women not taking contraceptive pills in corresponding age groups.     

Limnological studies of the lakes and streams of the upper qu'appelle river system,Saskatchewan, Canada

Summary Physical aspects including geography, geology, climate, soils, vegetation and lake morphometry of the upper Qu'Appelle River basin have been summarized. The importance of multiple use of this area has been stressed.The lakes were studied during the 1959–1969 period. Only Katepwa Lake showed temperature stratification for any length of time. Ice cover persisted for about five months each year. Buffalo Pound Lake had the lowest mean Secchi disc transparency (0.9 m) while Katepwa Lake had the highest (2.1 m). Light transmissibility tended to be lowest during May and September when wind velocities were highest. The 1 % level of light extinction was usually between 1 and 3 meters depth. The yellow portion of the spectrum penetrated the furthest.Buffalo Pound Lake was the least saline (600–700 mg/l TDS) while Last Mountain Lake was most saline (2000–2400 mg/l TDS). Input from Lake Diefenbaker and heavy spring runoffs effected major decreases in all lakes by May 1969. All lakes except Last Mountain were considerably more saline than during the 1937–1941 period. Variations in anions and cations within and between lakes vary with the total dissolved solids. As salinity increases magnesium, sulphate and chloride increase faster proportionally than other ions. The order of concentration of major cations is Na > Mg > Ca > K while the major anions' order of concentration is SO₄ > CO₃.HCO₃ > Cl.Soluble phosphate levels tend to be lowest in Buffalo Pound Lake. Levels increased sharply in Pasqua Lake below sewage outfalls from Moose jaw and Regina. Subsequently levels decreased in each successive downstream lake. Concentrations increased during the period of study. It was estimated that 594 metric tons of orthophosphate were contributed in domestic sewage in 1966. In addition land drainage contributes considerable amounts of phosphate during spring runoff.Nitrogen-bearing compounds vary in similar patterns as soluble phosphate but with smaller fluctuations. Domestic sewage and land drainage contribute significant amounts but of greater importance may be in situ biological fixation.All lakes were basic with usual pH of 8.4–9.0.From a chemical point of view these lakes are distinctly eutrophic.

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Zusammenfassung An fünf Seen des Ober Qu'Appelle Fluszgebietes, Saskatchewan, sind seit 1959 die jahreszeitlichen Veränderungen des Planktons und auch wichtige chemische und physikalische Milieufaktoren studiert worden. In diesem Teil werden die physiographischen Faktoren diskutiert.Dauernde Temperaturschichtung kommt nur im Katepwa See vor. Die 1% Lichtstärke war meistens zwischen 1 und 3 m Tiefe gelegen.Der Salzgehalt der Ober Qu'Appelle Seen liegt zwischen 600 mg/l (Buffalo Pound See) and 2400 mg/1 (Last Mountain See). Die Kationen ordnen sich in der Reihenfolge Na > Mg > Ca > K, die Anionen: SO₄ > CO₃.HCO₃ > Cl. Der pH bleibt meistens zwischen 8.4 and 9.0.Die Phosphatkonzentration ist sehr hoch. Abwasserverunreinigung, verursacht durch die Städte Regina und Moose jaw, ist bedeutsam für Phosphatgehalt in allen Seen auszer Buffalo Pound See. Die Stickstoffkonzentration ist ebenfalls hoch aber schwankt nicht soviel wie das Phosphat.Vom chemischen Standpunkt ausgesehen sind diese Seen sehr eutrophiert.

     

Chromatography of Microbial Lipids by Centrifugation Through Microparticulate Gel

An improved apparatus and a procedure are described by which the migration of sample components in column chromatography is accelerated by centrifugal force, thereby making it possible to use beds of densely packed gel prepared with ultrafine silica. This technique was used to resolve components of certain lipid mixtures where other methods have failed, and it has been found generally useful as an adjunct to other methods for the fractionation of lipids. Biologically active phosphoglycolipids from Mycobacterium tuberculosis and a phosphatidylglycerol-like substance from Mycoplasma pneumoniae which formed single spots on thin-layer chromatographic plates were each found to contain a major and several minor components by centrifugal chromatography. The method enabled us to isolate individual components of Wax D from M. tuberculosis rather than a spectrum of components. Minor components were resolved which, although present in insufficient quantity to influence results of chemical analyses, may be responsible for biological activity. The apparatus provides an essentially closed system which reduces highly volatile solvents to minimal evaporation during the chromatographic process. Samples are applied in solution and are not allowed to dry on the columns until after separation has been achieved. Consequently, polar, labile microbial lipids can be resolved without the use of harsh reagents which destroy some of their properties. Single components may be harvested by cutting and removing appropriate segments of the larger chromatograms or by eluting them from the columns.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.