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.     

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.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

A numerical taxonomic study of Propionibacterium strains from dairy sources

T.J. BRITZ AND K-H.J. RIEDEL. 1991. Phenotypic results from 81 tests conducted on 73 propionibacteria, including five type strains, 22 reference strains, unidentified propionibacteria and strains isolated from dairy sources, were analysed by numerical taxonomy. Characters giving uniform results were excluded. With the simple matching coefficient and the single linkage cluster analysis, 61 cultures were recovered in five major clusters. Final linkage of all the Propionibacterium cultures examined was at the 77% S-level. The results clearly showed that it was possible to distinguish between the 'classical' and 'cutaneous' Propionibacterium spp., corresponding with the type habitat of each group. The major 'classical' clusters were equated with the P. freudenreichii, P. thoenii, P. jensenii and P. acidipropionici species, while the only major 'cutaneous' cluster was equated with the P. acnes species. The major clusters were identified by relating them to specific type strains and by comparing phenotypic characteristics. The differentiating characteristics of each cluster were determined. The largest cluster, representing 37% of the strains, was equated with P. jensenii but contained cultures that produced an atypical brown/red pigment. These strains, although positively identified as P. jensenii , could also be identified as the 'old' P. rubrum ' species. Thus if pigmentation is used as differential characteristic two distinct groups of propionibacteria could be identified within the P. jensenii species.     

In vivo mechanical properties of leukocytes during adhesion to venular endothelium

Transient deformations of leukocytes (WBCs) were studied during their saltation along post-capillary venous endothelium (EC) in mesentery of the rat. During intermittent adhesion of WBCs to EC, prevailing fluid shear stresses, tau wall, resulted in a stepwise loading of the WBC upon attachment with a transient increase in length, L(t), and reduction in height, H(t). Measurements of L(t) and H(t) from frame-by-frame analysis of video recordings were modelled as the simple shear of a standard linear viscoelastic solid to facilitate calculation of the elastic (k1, k2) and viscous (mu) elements with k1 in parallel with serial elements k2 and mu. The magnitude of tau wall was determined from measurements of red cell velocity within the venule. During the spontaneous adhesion of WBCs, a value of cell viscosity (mu) of 45 Poise was determined. Stimulating adhesion by topical application of the chemoattractant FMLP resulted in a 15-fold increase of mu to 668 Poise. Transient deformations during topical application of cytochalesin B to disrupt actin fibers within the WBC, yielded a 40% reduction in k1, compared to an 80% reduction with colchicine which disrupts the microtubule structure. Thus, colchicine treated cells appear to be twice as deformable as cells treated with cytochalesin. During adhesion stimulated by the cytokine Interleukin-1, mu increased 50% without changes in k1 and k2, possibly due to slight activation of the WBC.