Human RNA “Rumor” Viruses: the Search for Novel Human Retroviruses in Chronic Disease

Summary: Retroviruses are an important group of pathogens that cause a variety of diseases in humans and animals. Four human retroviruses are currently known, including human immunodeficiency virus type 1, which causes AIDS, and human T-lymphotropic virus type 1, which causes cancer and inflammatory disease. For many years, there have been sporadic reports of additional human retroviral infections, particularly in cancer and other chronic diseases. Unfortunately, many of these putative viruses remain unproven and controversial, and some retrovirologists have dismissed them as merely “human rumor viruses.” Work in this field was last reviewed in depth in 1984, and since then, the molecular techniques available for identifying and characterizing retroviruses have improved enormously in sensitivity. The advent of PCR in particular has dramatically enhanced our ability to detect novel viral sequences in human tissues. However, DNA amplification techniques have also increased the potential for false-positive detection due to contamination. In addition, the presence of many families of human endogenous retroviruses (HERVs) within our DNA can obstruct attempts to identify and validate novel human retroviruses. Here, we aim to bring together the data on “novel” retroviral infections in humans by critically examining the evidence for those putative viruses that have been linked with disease and the likelihood that they represent genuine human infections. We provide a background to the field and a discussion of potential confounding factors along with some technical guidelines. In addition, some of the difficulties associated with obtaining formal proof of causation for common or ubiquitous agents such as HERVs are discussed.     

Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.     

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of Qs_rAb to .Abbreviations C concentration - D dilution rate - FBR fixed bed bioreactor - FIBR fluidised bed bioreactor - Gln glutamine - Qs cell specific rate - Qv volumetric rate - rAb recombinant antibody - X_v viable cell density - specific growth rate     

The effect of pH on feeding behaviour in newt larvae (Triturus: Amphibia)

The feeding responses of three species of newt larvae were compared under circumneutral and sublethal acid conditions. Under acid conditions (pH 4.5) feeding behaviour was suppressed in palmate newts, Triturus helveticus, and smooth newts. T. vulgaris , but not in crested newts, T. cristatus. At low pH, approach and orientation towards food occurred in T. helveticus and T. vulgaris , but snapping was inhibited; T. cristatus snapped and consumed food immediately it was offered under the same conditions. These differences are not consistent with the apparent greater tolerance of T helveticus for acidified ponds. The observations suggest that the chemosensory system of T. helveticus and T. vulgaris may be impaired at low pH.     

Identification and characterization of new DNA replication terminators in Bacillus subtilis

A functional DNA replication terminator of Bacillus subtilis contains two overlapping binding sites, A and B, for the replication terminator protein (RTP). A degenerate 17-mer oligonucleotide corresponding to the consensus B site has been used to detect four new terminators in the B. subtilis chromosome, in addition to the previously identified and closely spaced IRI and IRII. All the new terminators lie in the terminus region of the chromosome, on both sides of IRI and IRII, with their positions spanning <1O% of its length. Their DNA sequences are characterized by clearly identifiable A- and B-binding sites. They bind RTP in a manner indistinguishable from IRI, although precise affinities have not been compared. Each new terminator is functional in causing fork arrest when present in a plasmid replicating in B. subtilis . Three of the four were tested for polarity in fork-arrest activity and exhibited the polarity expected. The total of six terminators now identified in B. subtilis have been named TerI-TerVI . TerI and TerII correspond to the previously identified IRI and IRII, respectively. The chromosomal orientations of all but one of the terminators ( TerIV ) have been established and they conform to an arrangement similar to that in Escherichia coli in which two opposed groups of polar terminators provide a replication-fork trap ensuring that the approaching forks meet within a restricted region of the chromosome. The development of a strikingly similar arrangement of terminators in the two organisms, despite the lack of any detectable similarity in their respective DNA terminators and terminator proteins, emphasizes the importance of the replication-fork trap in each case.