Interrupting poliovirus transmission -- new solutions to an old problem.

Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, knowledge as to the nature of circulating polioviruses and the challenges to their interruption has increased tremendously, particularly during the period 2000-2005. By January 2006, however, the systematic application of the standard polio eradication strategies, combined with recent refinements, had reduced the number of countries with ongoing transmission of indigenous wild polioviruses to just four (Nigeria, India, Pakistan, and Afghanistan), the lowest ever in history. In addition, only 8 of the 22 areas that had been re-infected by wild poliovirus in 2003-2005 still required large-scale 'mop-up' activities and circulating vaccine-derived poliovirus (cVDPV) outbreaks were being readily addressed. This progress, despite new challenges late in the GPEI, was greatly facilitated by a range of solutions that included two new monovalent oral polio vaccines (mOPVs), new and robust international standards for polio outbreak response, and renewed political commitment across the remaining infected countries.     

Replication of damaged DNA

DNA damage is generated continually inside cells. In order to be able to replicate past damaged bases (translesion synthesis), the cell employs a series of specialised DNA polymerases, which singly or in combination, are able to bypass many different types of damage. The polymerases have similar structural domains to classical polymerases, but they have a more open structure to allow altered bases to fit into their active sites. Although not required for replication of undamaged DNA, some at least of these polymerases are located in replication factories. Emerging evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.     

Replication of damaged DNA: molecular defect in xeroderma pigmentosum variant cells.

Individuals with Xeroderma pigmentosum (XP) syndrome have a genetic predisposition to sunlight-induced skin cancer. Genetically different forms of XP have been identified by cell fusion. Cells of individuals expressing the classical form of XP (complementation groups A through G) are deficient in the nucleotide excision repair (NER) pathway. In contrast, the cells belonging to the variant class of XP (XPV) are NER-proficient and are only slightly more sensitive than normal cells to the killing action of UV light radiation. The XPV fibroblasts replicate damaged DNA generating abnormally short fragments either in vivo [A.R. Lehmann, The relationship between pyramidine dimers and replicating DNA in UV-irradiated human fibroblasts, Nucleic Acids Res. 7 (1979) 1901-1912; S.D. Park, J.E. Cleaver, Postreplication repair: question of its definition and possible alteration in Xeroderma pigmentosum cell strains, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 3927-3931.] or in vitro [S.M. Cordeiro, L.S. Zaritskaya, L.K. Price, W.K. Kaufmann, Replication fork bypass of a pyramidine dimer blocking leading strand DNA synthesis, J. Biol. Chem. 272 (1997) 13945-13954; D.L. Svoboda, L.P. Briley, J.M. Vos, Defective bypass replication of a leading strand cyclobutane thymine dimer in Xeroderma pigmentosum variant cell extracts, Cancer Res. 58 (1998) 2445-2448; I. Ensch-Simon, P.M. Burgers, J.S. Taylor, Bypass of a site-specific cis-syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts, Biochemistry 37 (1998) 8218-8226.], suggesting that in XPV cells, replication has an increased probability of being blocked at a lesion. Furthermore, extracts from XPV cells were found to be defective in translesion synthesis [A. Cordonnier, A.R. Lehmann, R.P.P. Fuchs, Impaired translesion synthesis in Xeroderma pigmentosum variant extracts, Mol. Cell. Biol. 19 (1999) 2206-2211.]. Recently, Masutani et al. [C. Masutani, M. Araki, A. Yamada, R. Kusomoto, T. Nogimori, T. Maekawa, S. Iwai, F. Hanaoka, Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity, EMBO J. 18 (1999) 3491-3501.] have shown that the XPV defect can be corrected by a novel human DNA polymerase, homologue to the yeast DNA polymerase eta, which is able to replicate past cyclobutane pyrimidine dimers in DNA templates. This review focuses on our current understanding of translesion synthesis in mammalian cells whose defect, unexpectedly, is responsible for the hypermutability of XPV cells and for the XPV pathology.     

Metabolic control: a new solution to an old problem

The phenomenon whereby the presence of oxygen regulates the rate of glucose metabolism was first described by Louis Pasteur. A novel mechanism has now been discovered, involving the AMP-activated protein kinase cascade, that can account for the Pasteur effect in ischaemic heart muscle.     

Evolution of genome size: new approaches to an old problem

Eukaryotic genomes come in a wide variety of sizes. Haploid DNA contents (C values) range > 80,000-fold without an apparent correlation with either the complexity of the organism or the number of genes. This puzzling observation, the C-value paradox, has remained a mystery for almost half a century, despite much progress in the elucidation of the structure and function of genomes. Here I argue that new approaches focussing on the genetic mechanisms that generate genome-size differences could shed much light on the evolution of genome size.     

Replication of DNA in mammalian chromosomes

A study of sedimentation and buoyant density of Okazaki fragments from mammalian chromosomes along with electron microscopic studies indicate that fragments from about 200 to 1200 nucleotides long may have RNA segments covalently attached. The fragments in some CsCl isopycnic gradients banded in two rather distinct bands. One band corresponds to the density of single-stranded DNA, but the other has a higher buoyant density which could be conferred by a segment of RNA up to 180 nucleotides or more in length. The RNA was not removed by denaturing conditions which separated DNA strands consisting of several thousand nucleotide pairs. When the material of higher buoyant density was spread for electron microscopy under conditions which would extend single-stranded DNA chains, but leave RNA in a coil or bush the chains with a higher buoyant density usually had a bush attached at one end. Under conditions that were thought to favor gap filling over chain elongation near growing forks, the DNA produced by pulse labeling with bromodeoxyuridine had a buoyant density which would indicate substitution to about 15 percent in one chain. If this substitution represents filling of gaps occupied by RNA before the pulse, the segments would be about 180 nucleotides in length assuming about 1,000 nucleotides between each segment.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Biofilms: new ideas for an old problem

Microbial cells, under moist conditions, are able to adhere to surfaces and to form structured communities embedded in a matrix of extracellular polymeric substances (EPS). In industrial environments, biofilms can cause heat and mass transfer limitations whilst in medical facilities they can be a source of contamination and proliferation of infections. Biofilm formation is related to the pathogenicity of some bacterial strains and cells in biofilms are usually resistant to antimicrobials agents, which increases the interest in new and sound methods for their prevention and destruction.     

Initiating the Uninitiated: Replication of damaged DNA and Carcinogenesis

Cancer is a genetic disease and carcinogenesis is the process whereby the relevant genetic alterations are acquired. Environmental carcinogens may damage DNA to induce mutations and chromosomal aberrations as permanent heritable changes in the genome that initiate carcinogenesis. For many carcinogens initiation of carcinogenesis requires the initiation of DNA replication suggesting that genetic alterations are fixed in the genome during replication of damaged DNA. It is of great interest to understand the mechanisms whereby carcinogen-induced damage to DNA causes mutations and chromosomal aberrations, and how cells may resist such events. It is clear now that cells express a complex repertoire of responses to DNA damage including several pathways of DNA repair and cell cycle checkpoints that protect against carcinogenesis. This commentary is concerned with the protective influence of DNA damage checkpoints that delay or arrest progression through the cell division cycle and especially with the responses of S phase cells to the environmental carcinogens UV and benzo[a]pyrene diolepoxide I (BPDE). Recent studies indicate that checkpoint responses may act at the very point of replication of damaged DNA to slow DNA chain elongation, inhibit replicon initiation, and suppress initiation of carcinogenesis.     

Manganese phytotoxicity: new light on an old problem

Background Manganese (Mn) is an essential micronutrient that is phytotoxic under certain edaphic and climatic conditions. Multiple edaphic factors regulate Mn redox status and therefore its phytoavailability, and multiple environmental factors including light intensity and temperature interact with Mn phytotoxicity. The complexity of these interactions coupled with substantial genetic variation in Mn tolerance have hampered the recognition of Mn toxcity as an important stress in many natural and agricultural systems.Scope Conflicting theories have been advanced regarding the mechanism of Mn phytotoxicity and tolerance. One line of evidence suggests that Mn toxicity ocurs in the leaf apoplast, while another suggests that toxicity occurs by disruption of photosynthetic electron flow in chloroplasts. These conflicting results may at least in part be attributed to the light regimes employed, with studies conducted under light intensities approximating natural sunlight showing evidence of photo-oxidative stress as a mechanism of toxicity. Excessive Mn competes with the transport and metabolism of other cationic metals, causing a range of induced nutrient deficiencies. Compartmentation, exclusion and detoxification mechanisms may all be involved in tolerance to excess Mn. The strong effects of light, temperature, precipitation and other climate variables on Mn phytoavailability and phytotoxicity suggest that global climate change is likely to exacerbate Mn toxicity in the future, which has largely escaped scientific attention.Conclusions Given that Mn is terrestrially ubiquitous, it is imperative that the heightened risk of Mn toxicity to both managed and natural plant ecosystems be factored into evaluation of the potential impacts of global climate change on vegetation. Large inter- and intraspecific genetic variation in tolerance to Mn toxicity suggests that increased Mn toxicity in natural ecosystems may drive changes in community composition, but that in agroecosystems crops may be developed with greater Mn tolerance. These topics deserve greater research attention.     

Replication initiation in mammalian cells: changing preferences

Accurate duplication of genetic material is central to cell proliferation. In eukaryotes, S phase is tightly controlled during development. For example, initiation events occur at random sites in early embryos but later appear restricted to preferred DNA regions. Epigenetic changes depending on chromatin organization and/or availability of specific factors likely control origin choice. By using the dynamic molecular combing technology, coupled with specific labeling of neo-synthesized DNA and FISH, we recently demonstrated that the efficiency and spacing of initiation sites are still flexible in mammalian somatic cells, and strongly rely on nucleotide availability. In all conditions, initiation events appear confined to short AT-rich sequences previously identified as matrix attachment regions, which suggests a direct involvement of these features in origin specification. Functional relationships between matrix anchorage and origin selection are discussed.     

Critical temperatures in lactating dairy cattle: A new approach to an old problem

Thermoregulatory reactions of lactating cows (33 Kg milk/day) have been measured in summer (T_g 25° – 39°C) and in winter (T_g 9, 5° – 24°C) at 3-hr intervals, during four 24-hr cycles. The animals maintained an almost continuous peripheral vasodilation throughout the experimental period. The upper ambient temperatures at which a dairy cow maintains homoeothermy were calculated for different metabolic rates. At the maintenance level a dry cow may maintain homoeothermy at up to 24°C without sweating and up to 40°C if sweating at 50% of the maximal sweating capacity. For a cow producing 30 kg milk/day, the respective figures were 12°C and 24°C respectively. These data indicate that the thermoregulatory capacity of the animals in the natural climate considerably expands the thermal comfort temperature range.

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Zusammenfassung Die wärmeregulatorischen Reaktionen milchgebender Kühe (33 kg Milch/Tag) wurden im Sommer (T_g 25° – 39°C) und im Winter (T_g 9, 5° – 24°C) in 3-Stunden Intervallen während 4 mal 24 Stunden periodisch gemessen. Die Tiere hatten eine beinahe kontinuierliche periphere Gefässerweiterung während der Versuchsperioden. Die obere Umgebungstemperatur, bei der Milchkühe homoiothermisch bleiben, wurde aus verschiedenen Stoffwechselraten berechnet. Auf dem Erhaltungsniveau kann eine trocken stehende Kuh die Homoiothermie bis 24°C ohne Schwitzen halten und bis auf 40°C bei Schwitzen bis 50% des maximalen Schwitzvermögens. Für eine Kuh, die 30 kg Milch/Tag leistet sind die entsprechenden Werte 12°C beziehungsweise 24°C. Diese Werte zeigen, dass das Wärmeregulationsvermögen der Tiere im natürlichen Klima den Wärmekomfort-Temperaturbereich erheblich ausdehnt.

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Resume On a mesuré l'aptitude de thermorégulation de vaches en lactation (33 kg de lait par jour) toutes les 3 heures durant 4 cycles de 24 heures et cela aussi bien en été (T_g 25° à 39°C) qu'en hiver (T_g 9, 5° à 24°C). Les animaux ont présenté une vasodilatation périphérique assez constante durant toute la période des essais. On a en outre calculé, en partant de différents taux de métabolisme, la température ambiante maximum pour laquelle une vache laitière peut maintenir son homéothermie. Pour une vache sèche, ce maximum est de 24°C sans transpirer et de 40°C si elle transpire le 50% de sa capacité maximum. Pour une vache donnant 30 kg de lait par jour, ces chiffres s'abaissent à 12°C dans le premier cas, à 24°C dans le second. Ces chiffres montrent que la capacité de thermorégulation du bétail bovin augmente considérablement l'amplitude de la zone de confort thermique en climat naturel.

     

Replication of kinetoplast DNA: an update for the new millennium

In this review we will describe the replication of kinetoplast DNA, a subject that our lab has studied for many years. Our knowledge of kinetoplast DNA replication has depended mostly upon the investigation of the biochemical properties and intramitochondrial localisation of replication proteins and enzymes as well as a study of the structure and dynamics of kinetoplast DNA replication intermediates. We will first review the properties of the characterised kinetoplast DNA replication proteins and then describe our current model for kinetoplast DNA replication.     

Using new tools to solve an old problem: the evolution of endothermy in vertebrates

During the past 30 years, the evolution of endothermy has been a topic of keen interest to palaeontologists and evolutionary physiologists. While palaeontologists have found abundant Permian and Triassic fossils, suggesting important clues regarding the timing of origin of endothermy, physiologists have proposed several plausible hypotheses of how the metabolic elevation leading to endothermy could have occurred. More recently, molecular biologists have developed powerful tools to infer past adaptive processes, and gene expression mechanisms that describe the organization of genomes into phenotypes. Here, we argue that the evolution of endothermy could now be elucidated based on a joint, and perhaps unprecedented, effort of researchers from the fields of genomics, physiology and evolution.