ProMED global monitoring of emerging diseases: design for a demonstration program.

The emergence and reemergence of infectious diseases, as the result of recent and ongoing social and environmental changes, urgently calls for a global surveillance system, so that unusual outbreaks can be recognized and controlled at an early stage. ProMED, an international non-governmental group of infectious disease experts, was organized by the Federation of American Scientists to promote the establishment of a global Program to Monitor Emerging Diseases. ProMED proposes the establishment of a demonstration program by prioritizing a small number of strategically-located institutions in the developing world, mainly those least in need of upgrading, for development as sentinel centers. In this way a functional, although limited, network with capabilities for monitoring both endemic and emerging diseases could be rapidly established at minimal cost. The network would serve as an experimental model for future expansion. Initially, each center would develop its own local/regional network with which it would exchange information and assistance, and through which it would collect clinical data and specimens for monitoring the emergence of a limited number of defined syndromes. A central program office would provide protocols, assistance, training, quality assurance, communications, etc. and would coordinate fundraising and program activities. If successful, the syndromes monitored would be expanded and additional institutions strengthened to become new network centers.

Biological defense research: charting a safer course.

KIE: Jacobson and Rosenberg comment on aspects of the U.S. Army's Biological Defense Research Program and propose shifting some responsibility for biological defense research to a civilian agency such as the National Institutes of Health. They call for more openness in the conduct of such research, and report on Congressional efforts to reinforce the 1972 Biological Weapons Convention. Finally, they suggest ways in which physicians can be sure that research on biological agents is conducted safely and appropriately and can work to decrease the threat of a biological arms race.^ieng

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes.

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.

Inhibition of DNA synthesis, independent of DNA adduct formation, by benzo[a]pyrene diol epoxide in mammalian cells.

Treatment of SV40-infected CV-1 cells with the ultimate carcinogen anti-benzo[a]pyrene diol epoxide (BPDE) at 1 X 10(-4) mg/ml or higher reduced the rate of viral DNA synthesis to an extent dependent on the BPDE concentration; similar reductions in cellular DNA synthesis were produced in infected and uninfected cells. Treatment of cells with BPDE, followed by removal of BPDE, at various times before infection with SV40 gave the same results. Recovery or partial recovery of DNA synthesis occurred when the BPDE concentration was below 6 X 10(-4) mg/ml; at higher concentrations the cells were killed. Simultaneously replicating viral DNA's from viruses infecting the same cells before and after BPDE treatment of the cells exhibited the same reduced rate of synthesis. The evidence indicates that covalent adducts in viral DNA are not responsible for its reduced replication rate; moreover, it is probable that an insignificant number of adducts is produced in intracellular viral DNA at BPDE concentrations that do not kill CV-1 cells. Rather, it appears likely that BPDE inhibits viral DNA synthesis by attacking cellular DNA or non-DNA targets. Caution is therefore required in relating the effects of BPDE and other carcinogens to DNA adduct formation.

Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts on the template.

The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phiX174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough.

Effects of benzo[a]pyrene diol epoxide adducts on DNA synthesis in mammalian cells.

The replication of DNA containing anti-benzo[a]pyrene diol epoxide (BPDE) adducts was studied in mammalian cells by first treating SV40 virus with BPDE in vitro, then infecting cells with virus containing a known number of adducts in the DNA. Viral transcription products necessary for replication were supplied by co-infection with an untreated virus containing a deletion as a DNA marker. Thus, only replicative effects of BPDE adducts were manifested. Delayed replication of the DNA from BPDE-treated virus, relative to the DNA containing the deletion, was observed, but in time most or all of the infecting molecules were able to replicate. The results are consistent with the hypothesis that adducts of BPDE in DNA block DNA synthesis in vivo, as they do in vitro, and that the block is gradually overcome by a repair mechanism that eliminates the adducts responsible for blockage or by delayed replicative bypass of the adducts. In spite of the ability of the system to overcome the delay in replication, the viability of the BPDE-treated virus in plaque assay was low, suggesting a persistent defect in transcription or a high level of error in repair or bypass replication.

Prereplicative events involving simian virus 40 DNA in permissive cells.

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA in the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TAqI restriction enzyme was used to provide viral functions required for replication, and a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.

Growth and purification of SV40 virus for biochemical studies.

A detailed growth and purification scheme suitable for producing relatively large quantities of fully active, pure SV40 is presented together with data on recovery and purity at each step of the procedure. The scheme was designed to prevent the initial binding of virus to cell components as well as contamination of the extracted virus by cellular DNA.

The major adducts of cis and trans benzo[a]pyrene diol epoxides cause chain termination during DNA synthesis in vitro.

We have studied DNA synthesis in vitro using as template phi X174 DNA containing varying numbers of adducts formed by reaction with cis and trans benzo[a]pyrene (BP) diol-epoxides. The extent of DNA synthesis decreases with increasing numbers of adducts and there is a concomitant decrease in the size of the DNA products. Both decreases can be accounted for quantitatively by the assumption that synthesis terminates at every BP adduct. Since the majority of the adducts are located at the 2-amino group of guanine, we deduce that these adducts cause termination. The role of adducts at other sites is uncertain. The cis and trans BP diol-epoxides are indistinguishable with regard to chain termination, yet in vivo these isomers behave differently. These results suggest that chain determination alone is insufficient to account for the mutagenic effects of BP diol-epoxides.

Effects of ethylating agents on DNA synthesis in vitro: implications for the mechanism of carcinogenesis.

A highly carcinogenic ethylating agent, ethylnitrosourea (ENU), and a weakly carcinogenic one, diethylsulfate (DES) react with DNA to roughly the same extent but DES produces about 6 times as many unstable ethylated bases, which are gradually lost spontaneously under physiological conditions. The different rates of loss for the different DNA bases have been studied using polydeoxyribonucleotides. Spontaneous strand breakage following base loss is slow, lagging more than a week behind base loss at 37 degrees C; ultimately, DES results in much more spontaneous strand breakage than ENU. In DNA synthesis in vitro, using avian myeloblastosis virus (AMV) polymerase, no nucleotides are incorporated opposite missing bases in the template; when the template contains ethylated bases that are impaired in their ability to form specific hydrogen bonds, purine-pyrimidine mispairing can occur and mismatched nucleotides are incorporated into the daughter strand. ENU ethylates somewhat more sites leading to mispairing potential than DES. ENU also produces approximately 7.5 times more ethyl phosphotriesters than DES.

DNA swivel enzyme activity in a nuclear membrane fraction.

DNA swivel (nicking-rejoining) enzyme activity has been studied in various cell fractions of a human lymphoid cell line. Swivel activity is found only in chromatin and in a nuclear membrane fraction containing DNA and possessing endogenous DNA synthesizing activity. Twenty percent of the total swivel activity and less than one percent of the total DNA are in the membrane fraction. The swivel enzyme is more tightly bound to the membrane fraction than to the chromatin fraction. These observations suggest that the swivel enzyme may be a replication factor, specifically bound to replicating DNA in the membrane fraction.

Variation in DNA swivel enzyme activity during the mammalian cell cycle.

Synchronized cells of a normal human lymphocytic cell line contain little swiven enzyme activity in G0 and G1 and high activity in Sphase. The level of activity in different growth phase appears to be related to the fraction of the population engaged in DNA replication. No endogenous inhibitor or activator of swiven activity could be demonstrated. The evidence implies that the enzyme may be present only during S phase; it is therefore a possible control factor for replication.

The negative control mechanism for E. coli DNA replication.

Evidence is presented to show that the initiation of DNA replication in E. coli 555-7 requires synthesis of a protein whose production is correlated with total protein synthesis. Once replication is initiated, however, reinitiation will occur if all further protein synthesis is prevented; a small amount of protein synthesis is sufficient to prevent this unregulated reinitiation. This shows that the initiation of DNA replication is under negative control. A mechanism for the control of DNA replication is proposed; in this mechanism a replication repressor is synthesized periodically, while an antirepressor protein is synthesized continuously. Derepression of initiation results after sufficient accumulation of the antirepressor protein, and repression is re-established by repressor synthesis after the initiation of replication.

Shear and the melting of DNA: an especially sensitive portion of the E. coli genome.

The melting point of DNA is shown to be a function of shear stress. The higher the molecular weight of the DNA, the further its melting point is lowered by a given shear rate. During lysis of E. coli, a part of the DNA is especially shear sensitive, so that its melting curve in the presence of shear shows a low-melting region prior to the main transition. Lysis and dilution of the cell contents destroys the extra shear sensitivity, perhaps because the DNA dissociates from the cell membrane or from some other large subcellular structure. Such a structure would impart increased shear sensitivity to the associated region of the genome.