The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Naul. Acad. Sci. USA 82,[7096][7097][7098][7099][7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, -di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di-and triphosphate were low (c5 MuM and <2 MuM, respectively).Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent K. values of 2.9MuM and 3.0 ,uM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 ,uM vs. 4.1 MM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. ICso (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HYIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase a. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase a. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED50) = 0.1 ,uM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virusspecified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di-and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine.The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsAl).
The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2" over Mg2".
Nachhaltigkeit ist zugleich historisches Kulturgut und moderne Antwort auf die aktuelle globale Krisensituation im Kontext des Klimawandels. Insbesondere im Bauwesen ist der strategische Ansatz der Nachhaltigkeit in den letzten Jahren durch die Entwicklung und Implementierung von Nachhaltigkeitszertifizierungssystemen fest verankert worden. Derzeit weist die Bau‐ und Immobilienwirtschaft vor allem in den Bereichen der Diversifikation entwickelter Bewertungs‐ und Optimierungswerkzeuge und der Ausbildung eines praxistauglichen rechtlichen Rahmens für die beteiligten Akteure eine wesentliche Dynamik auf. Beyond Platin – Sustainability trends in the construction and real estate industry. Sustainability is both a cultural asset and a modern answer to the current global crisis of climate change. In the past few years, sustainability has become especially important in the construction industry, anchored by the development and implementation of a number of sustainability certification systems. Today, the construction and real estate industry shows an essential dynamism in areas of diversifying of the developed assessment and optimization tools as well as establishing of a suitable legal framework for the involved players.
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