A dnaB-like protein of Pseudomonas aeruginosa.

A dnaB-like protein from P. aeruginosa was purified to near homogeneity using as an assay the immunoprecipitation by E. coli dnaB antiserum in a solid-phase. In the chromatographic characteristics including the affinity to immobilized ATP the dnaB-like protein of P. aeruginosa is similar to the dnaB protein of E. coli with the exception that it does not bind to heparin-Sepharose. The dnaB-like protein has a native molecular weight of about 320,000 as determined by glycerol gradient sedimentation. It consists of several identical subunits of molecular weight of 56,000 as measured in a denaturing SDS gel. Associated with the enzyme is a DNA-dependent ATPase- and helicase activity. The dnaB-like protein is similar to the E. coli dnaB protein with regard to the binding of ATP gamma S and the formation of a ternary complex consisting of the enzyme, ATP gamma S, and phi X174 DNA. However, the enzyme of P. aeruginosa is inactive in a phi X174 DNA-dependent in vitro dnaB complementation assay using an E. coli dnaBts extract.     

Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma.

We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week-old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S.     

Reactions in vitro of the DNA polymerase-primase from Xenopus laevis eggs. A role for ATP in chain elongation

A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.     

Pectinases in Aqueous Extracts of Ditylenchus dipsaci

Aqueous extracts of a population of Ditylenchus dipsaci isolated from onion and maintained monoxenically on onion callus contained endo-polygalacturonase (endo-PG) and endo-pectinmethyltranseliminase (endo-PMTE). In viscometric tests pH 4.2 and 4.0 were optimal for degradation of sodium polypectate and pectin N.F., respectively, by endo-PG. Endo-PMTE reduced viscosity of pectin N.F. optimally at pH 8.5 or above. Activity was dependent on CaCl₂. Pectinmethylesterase activity was not detected in water, NaCl, or sucrose extracts of these nematodes. The extracts macerated potato tuber tissue, onion cotyledonary tissue, and strips of onion epidermis from the ventral surface of onion bulb scales at pH 4.2, 5.3, and 6.2. Pectin could not be localized with hydroxylamine-ferric chloride reagent in macerated tissues treated for 24 hr with active extract.     

A Comparison of Pectinases from Ditylenchus dipsaci and Allium cepa Callus Tissue

Ground and whole Ditylenchus dipsaci maintained on onion callus contain no culturable micro-organisms when tested with five check media. Healthy onion callus does not produce pectolytic enzymes. Pectolytic enzymes are present in infected callus. These enzymes are, however, associated with resident nematodes and not host tissues. These results suggest that D. dipsaci is the actual source of the endo-polygalacturonase and endo-pectinmethyltrans-eliminase extracted from them.     

Organstruktur und Zellfunktion in explantiertem und später auto-retransplantiertem Schilddrüsengewebe

Zusammenfassung Schilddrüsengewebe von erwachsenen Kaninchen wurde in heterologem Medium in Rollröhrchen gezüchtet und 3 Wochen bis 4 1/2 Monate später auf das Spenderkaninchen rückverpflanzt. Hier blieben die Transplantate von 1 bis zu 8 Monaten. Die histologischen Befunde unmittelbar vor und nach Abschluß der Transplantation wurden miteinander verglichen. Unter beiden Lebensbedingungen, in vitro und im Transplantat, produzierten die Schilddrüsenzellen massenhaft Sekret, das in seinem färberischen Verhalten dem Schilddrüsenkolloid gleicht. Es wird für unwahrscheinlich gehalten, daß das Sekret biochemisch vollwertiges Schilddrüsenkolloid darstellt. Das Sekret wurde in großen Mengen intrazellulär gestapelt und führte schließlich zum Zellverfall.In den Transplantaten bestand nur dann Follikelanordnung, wenn diese auch noch in der Ausgangskultur vorhanden war. Die Follikel waren in jungen Transplantaten zunächst recht gut von Kapillaren umsponnen, doch verödeten diese später wieder. Alte Transplantate gingen schließlich genau so wie die gefäßlosen Gewebekulturen und wie alte, von vorn herein gefäßlos bleibende Transplantate durch intrazelluläre Sekretstapelung zugrunde. Es wird vermutet, daß in vitro der Mangel an thyreotropem Hormon zu dieser Fehlsteuerung führte und daß die Zellen auch im Transplantat nicht mehr auf das nun zur Verfügung stehende thyreotrope Hormon mit Ausschleusung des Sekrets reagieren konnten, weil dieser Mechanismus schon vorher in vitro pathologisch verändert worden war.Durch die Vorzüchtung wurde das Autotransplantationsergebnis erheblich verschlechtert.Eine maligne Entartung trat während der Züchtung in vitro nicht ein.     

Die Ultrastruktur des Juxtaglomerulären Apparates des Meerschweinchens

Zusammenfassung Der Beweis für das Vorkommen von Sekretgranula in den epitheloiden Zellen des Meerschweinchens ist bisher von keinem Autor erbracht worden. Ihre Abwesenheit ist um so erstaunlicher, als die Renin-Aktivität in der Niere dieses Tieres etwa 1/10 der der Ratte mit ihren stark granulierten Epitheloidzellen beträgt und die des Menschen sogar übertrifft. In unserem Untersuchungsgut finden wir hin und wieder einige wenige, wahrscheinlich Renin enthaltende Granula (Abb. 1–4). Anscheinend erfolgen Synthese und Ausscheidung des Enzyms im gleichen Rhythmus; zu einer geringgradigen Speicherung kommt es offenbar nur unter bestimmten funktionellen Bedingungen.Die Goormaghtighschen Zellen (Abb. 5) zeigen kein besonderes auffälliges artspezifisches Verhalten.An der Macula densa wird erstmals eine starke Erweiterung sowohl der intracytoplasmatischen Einfaltungen des basalen Plasmalemms — des basalen Labyrinthes — als auch der Interzellularspalten beschrieben (Abb. 6–9). Diese oberhalb der Basalmembran gelegenen Pseudovakuolen sind somit extrazellulär und möglicherweise als morphologisches Äquivalent einer starken Reabsorptionstätigkeit zu deuten. Es ist zur Zeit noch nicht entschieden, ob sie mit der von uns angewandten Präparationstechnik auch bei anderen Tieren und beim Menschen darstellbar sind.

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Summary The presence of secretory granules in the epithelioid (juxtaglomerular) cells in the media of the preglomerular portion of the afferent arterioles in the kidney of the Guinea pig has not been proven by any author until now. The absence of these granules is all the more astonishing in view of the fact that the renin activity of this animal is about 1/10 of that of the rat — with its highly granulated cells — and even surpasses that of humans. In our inbread strain rare granules likely containing renin can be found from time to time (Fig. 1–4). Apparently, the synthesis and extrusion of the enzyme takes place with the same rhythm; a very low degree of accumulation occurs probably only under certain functional conditions.The Goormaghtigh Cells (Fig. 5) show no noticeable differences specific for the Guinea pig. In the Macula densa highly developed enlargements of the basal infoldings of the plasma membrane — basal labyrinthe — as well as of the intercellular spaces are described for the first time (Fig. 6–9). These enlargements (Pseudovacuoles), situated above the basement membrane, are extracellular and can possibly be interpreted as the morphological equivalent of a high degree of absorption activity. At the present time it has not been decided, if, using the authors' technique, these enlargements can be demonstrated in other animals and humans.

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In Zusammenarbeit mit dem Pharmakologischen Institut der Universität Lausanne und mit Unterstützung durch die Fritz Hoffmann-La Roche-Stiftung zur Förderung wissenschaftlicher Arbeitsgemeinschaften in der Schweiz.     

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.