Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Widespread Infection with Homologues of Human Parvoviruses B19, PARV4, and Human Bocavirus of Chimpanzees and Gorillas in the Wild

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.Autonomous parvoviruses known to infect humans comprise parvovirus B19 (18) and the recently discovered PARV4 (22) and human bocavirus (HBoV) (3). Members of the family Parvoviridae are genetically and biologically diverse and are classified into several genera or groups, showing marked differences in host range, pathology, and tissue/cellular tropisms (18). Human parvovirus B19, a member of the Erythrovirus genus, is transmitted primarily by the respiratory route but causes systemic infections. Erythroid progenitor cells are specifically targeted through expression of globoside P antigen, which acts as the B19 virus receptor for entry (5). In common with infections by most parvoviruses, B19 virus infections are acute; a period of intense viremia is followed by seroconversion for antibody to B19 virus and lifelong immunity from reinfection (29). Despite the clearance of viremia and seroconversion for antibody, lifelong persistence of viral DNA in tissues has been shown to occur (12, 20, 26, 28, 43, 58). Three genotypes of B19 virus have been described, differing in nucleotide sequence by approximately 13 to 14% (7, 21, 41, 53); genotypes 1 and 2 have been found in Europe, the United States, and other Western countries, while genotype 3 is restricted to sub-Saharan Africa and South America (7, 47, 49). B19 virus widely circulates in human populations worldwide; in Western countries, several studies have documented increasing frequencies of B19 virus seropositivity with age, rising to approximately 60 to 70% by adulthood (15, 39, 48, 61).Another human parvovirus, PARV4, shows markedly different epidemiology and transmission routes. It was originally detected in plasma from an individual with an “acute infection syndrome” resembling that of primary human immunodeficiency virus (HIV) infection (22). While this clinical presentation has not been observed again, infection with PARV4 is known to be widespread specifically in individuals with a history of parenteral exposure (injecting drug users [IDUs], hemophiliacs, polytransfused individuals), with a strikingly higher incidence in those infected with HIV-1 (13, 14, 30, 35, 54). These observations suggest that PARV4 is primarily transmitted though parenteral routes in Western countries (54, 56). In common with infection with the better-characterized human parvovirus B19, infection with PARV4 is associated with a period of acute viremia, followed by seroconversion for antibody and long-term persistence of viral DNA sequences in lymphoid and other tissue (33, 37, 52). Circulating variants of PARV4 have been classified into three distinct genotypes exhibiting approximately 8% nucleotide sequence divergence from each other. Genotypes 1 and 2 circulate in Western countries, while genotype 3 has to date been recorded only in sub-Saharan Africa (45, 55).The third human parvovirus, HBoV (3), shows a number of epidemiological and clinical attributes different from those of both B19 virus and PARV4. HBoV was originally found in the respiratory tract of young children and has been the subject of intense investigation as a potential cause of human respiratory disease (reviewed in references 1, 51, and 62). Although it is frequently detected by PCR in the nasopharynx of viremic individuals with primary infections with lower respiratory tract disease, other coinfecting respiratory viruses are frequently detected (19). HBoV additionally shows long-term, low-level carriage in the respiratory tract after primary infection, which further complicates PCR-based etiological studies (2, 38) and warrants the use of other diagnostic strategies, such as serology (30, 32, 59). In contrast to the rather minimal genetic diversity of B19 virus and PARV4 genotypes, bocaviruses infecting humans are now known to comprise three to four major genetic variants (termed types or species 1 to 4) (23, 24). HBoV1 and HBoV2 show 22%, 33%, and 20% amino acid sequence divergence from each other in the encoded viral nonstructural (NS), NP-1, and structural VP1/VP2 proteins, respectively, the latter potentially leading to antigenic diversity and some loss of antigenic cross-reactivity. A third type/species of HBoV is a chimeric form with a nonstructural gene region (NS, NP1) most similar to HBoV1, a recombination breakpoint in the intergenic region between NP1 and VP1, and structural genes related to those of HBoV2 (4, 23). Current data suggest that only HBoV1 is capable of infecting the respiratory tract; most published large-scale screening studies have failed to detect HBoV2 (or HBoV3) in respiratory samples (10, 11, 60), while all three types/species are detectable in fecal samples, indicating the existence of an alternative or additional site of virus replication (23). Despite extensive inquiry, the exact role of HBoV1 in respiratory disease remains unclear, as is the proposed etiological role of HBoV2 (and possibly HBoV3) in gastroenteritis (4, 11, 23, 50). Very recently, a fourth species/type, HBoV4, has been detected in fecal samples; genetically it also shows evidence for past recombination, with NS and NP1 region sequences grouping with HBoV2, while VP1/VP2 is more closely related to HBoV3 (23).We have little understanding of the past epidemiology, evolution, and origins of human parvoviruses. For both B19 virus and PARV4, evidence has been obtained for a temporal succession of genotypes over time (37, 43); in Europe, B19 virus genotype 1 largely replaced type 2 in the 1960 and 1970s (43), while current data indicate that a similar replacement of PARV4 genotypes occurred within the last 20 years (37). The highly restricted sequence diversity of currently circulating variants of PARV4 and B19 virus and of HBoV1 variants supports the hypothesis of a relatively recent emergence and spread of these viruses in human populations (36, 42, 64).The existence and evolution of parvoviruses on a much longer time scale is suggested by the observations that members of the Erythrovirus and Parvovirus genera both contain viruses that are highly host species specific and that the molecular phylogenies of both genera are largely congruent with those of their hosts (34). This has led to the hypothesis of long-term coevolution of parvoviruses with their host over the 90 million years of mammalian evolution and perhaps beyond. Among erythroviruses, simian homologues of B19 virus have been found in cynomolgus monkeys (44) and rhesus and pig-tailed macaques (16) and more genetically distant viruses have been characterized in chipmunks and cows (9, 63). Divergent homologues of PARV4 in pigs and cows have been described (31), while the bovine and canine parvoviruses distantly related to HBoV are the originally described members of the Bocavirus genus. However, the process of virus-host codivergence is known to be punctuated by occasional cross-species transmissions, including the well-documented spread of feline parvovirus to dogs (46). Based on serological evidence, the possible transmission of simian erythroviruses to animal handlers has been proposed (6).To gain further insights into the origins and evolution of human parvoviruses, we have performed large-scale serological and PCR-based screening of nonhuman primates (chimpanzees and gorillas) and of several species of Old World monkeys (OWMs) for evidence of infection with parvoviruses that are antigenically related to the human B19, PARV4, and HBoV viruses. By PCR, we have sought to genetically characterize homologues of the three autonomous human parvoviruses in apes and Old World monkey species and to analyze their evolutionary relationship to human and other mammalian homologues of these viruses.     

Patterns of bushmeat hunting and perceptions of disease risk among central African communities

There is a great need to determine the factors that influence the hunting, butchering and eating of bushmeat to better manage the important social, public health and conservation consequences of these activities. In particular, the hunting and butchering of wild animals can lead to the transmission of diseases that have potentially serious consequences for exposed people and their communities. Comprehension of these risks may lead to decreased levels of these activities. To investigate these issues, 3971 questionnaires were completed to examine the determinants of the hunting, butchering and eating of wild animals and perceptions of disease risk in 17 rural central African villages. A high proportion of individuals reported perceiving a risk of disease infection with bushmeat contact. Individuals who perceived risk were significantly less likely to butcher wild animals than those who perceived no risk. However, perception of risk was not associated with hunting and eating bushmeat (activities that, compared with butchering, involve less contact with raw blood and body fluids). This suggests that some individuals may act on perceived risk to avoid higher risk activity. These findings reinforce the notion that conservation programs in rural villages in central Africa should include health-risk education. This has the potential to reduce the levels of use of wild animals, particularly of certain endangered species (e.g. many non-human primates) that pose a particular risk to human health. However, as the use of wild game is likely to continue, people should be encouraged to undertake hunting and butchering more safely for their own and their community's health.     

Extinct,obscure or imaginary: The lizard species with the smallest ranges

Aim

Small geographic ranges make species especially prone to extinction from anthropogenic disturbances or natural stochastic events. We assemble and analyse a comprehensive dataset of all the world's lizard species and identify the species with the smallest ranges—those known only from their type localities. We compare them to wide‐ranging species to infer whether specific geographic regions or biological traits predispose species to have small ranges.

Location

Global.

Methods

We extensively surveyed museum collections, the primary literature and our own field records to identify all the species of lizards with a maximum linear geographic extent of <10 km. We compared their biogeography, key biological traits and threat status to those of all other lizards.

Results

One in seven lizards (927 of the 6,568 currently recognized species) are known only from their type localities. These include 213 species known only from a single specimen. Compared to more wide‐ranging taxa, they mostly inhabit relatively inaccessible regions at lower, mostly tropical, latitudes. Surprisingly, we found that burrowing lifestyle is a relatively unimportant driver of small range size. Geckos are especially prone to having tiny ranges, and skinks dominate lists of such species not seen for over 50 years, as well as of species known only from their holotype. Two‐thirds of these species have no IUCN assessments, and at least 20 are extinct.

Main conclusions

Fourteen per cent of lizard diversity is restricted to a single location, often in inaccessible regions. These species are elusive, usually poorly known and little studied. Many face severe extinction risk, but current knowledge is inadequate to properly assess this for all of them. We recommend that such species become the focus of taxonomic, ecological and survey efforts.

     

Ecophysiological variation across a forest‐ecotone gradient produces divergent climate change vulnerability within species

Climate change related risks and impacts on ectotherms will be mediated by habitats and their influence on local thermal environments. While many studies have documented morphological and genetic aspects of niche divergence across habitats, few have examined thermal performance across such gradients and directly linked this variation to contemporary climate change impacts. In this study, we quantified variation in thermal performance across a gradient from forest to gallery forest‐savanna mosaic in Cameroon for a skink species (Trachylepis affinis) known to be diverging genetically and morphologically across that habitat gradient. Based on these results, we then applied a mechanistic modelling approach (NicheMapR) to project changes in potential activity, as constrained by thermal performance, in response to climate change. As a complimentary approach, we also compared mechanistic projections with climate‐driven changes in habitat suitability based on species distribution models of forest and ecotone skinks. We found that ecotone skinks may benefit from warming and experience increased activity while forest skinks will likely face a drastic decrease in thermal suitability across the forest zone. Species distribution models projected that thermal suitability for forest skinks in coastal forests would decline but in other parts of the forest zone skinks are projected to experience increased thermal suitability. The results here highlight the utility of mechanistic approaches in revealing and understanding patterns of climate change vulnerability which may not be detected with species distribution models alone. This study also emphasizes the importance of intra‐specific physiological variation, and habitat‐specific thermal performance relationships in particular, in determining warming responses.     

UV photoelectron spectroscopy and ab initio characterization of valence orbital structures and conformations of neutral phosphate esters

The HeI UV photoelectron spectrum of trimethyl phosphate (TMP) has been measured and interpreted with the aid of SCF molecular orbital calculations carried out with STO-3G, STO-3G* and 4-31G basis functions. The photoelectron spectrum of TMP is more accurately reproduced by results from 4-31G calculations than by results from STO-3G or STO-3G* calculations. However, all three basis sets yield results which predict the same assignment of the photoelectron spectrum. Results at the 4-31G level indicate that whether calculations are based on crystallographic bond angles and bond lengths or on STO-3G optimized geometries has little effect on the energetic ordering of the upper occupied orbitals. The energetic ordering of orbitals is also found to be only weakly dependent upon the torsional angle phi, describing rotation of ester groups about P-O bonds and upon the torsional angle psi, describing rotation of methyl groups about C-O bonds. For trimethyl phosphate, with C3 symmetry, the vertical ionization potentials of the upper occupied orbitals are 10.81 eV (8e), 11.4 eV (9a), 11.93 eV (7e), 12.6-12.9 eV (8a and 6e), 14.4 eV (7a) and 15.0-16.0 eV (5e and 6a). Calculations at the 4-31G level indicate that many of the highest occupied orbitals in neutral dimethyl phosphate and methyl phosphate have energies and electron distributions similar to orbitals in TMP. For TMP, a search for optimized values of phi and psi has been carried out at the STO-3G*level. In agreement with previous NMR studies and with classical potential calculations, the STO-3G* results indicate that both the gauche (phi = 53.1 degrees) and anticlinal (phi = 141.9 degrees) conformations are thermally accessible. Also in agreement with the classical potential calculations, the STO-3G* results predict that in the all gauche conformation energy is minimized when the methyl groups assume a staggered geometry (psi = 60 degrees to 80 degrees) and that an energy maximum occurs for an eclipsed geometry (phi = 0 degrees to 20 degrees). A study of the dependence of optimized values of O-P-O ester bond angles on the torsional angles, phi, was carried out at the STO-3G, STO-3G* and 4-31G levels. The results demonstrate that for C3 symmetry, the coupling of O-P-O angles to phi is influence by repulsive steric interactions.     

Pathogen-Host Associations and Predicted Range Shifts of Human Monkeypox in Response to Climate Change in Central Africa

Climate change is predicted to result in changes in the geographic ranges and local prevalence of infectious diseases, either through direct effects on the pathogen, or indirectly through range shifts in vector and reservoir species. To better understand the occurrence of monkeypox virus (MPXV), an emerging Orthopoxvirus in humans, under contemporary and future climate conditions, we used ecological niche modeling techniques in conjunction with climate and remote-sensing variables. We first created spatially explicit probability distributions of its candidate reservoir species in Africa''s Congo Basin. Reservoir species distributions were subsequently used to model current and projected future distributions of human monkeypox (MPX). Results indicate that forest clearing and climate are significant driving factors of the transmission of MPX from wildlife to humans under current climate conditions. Models under contemporary climate conditions performed well, as indicated by high values for the area under the receiver operator curve (AUC), and tests on spatially randomly and non-randomly omitted test data. Future projections were made on IPCC 4^th Assessment climate change scenarios for 2050 and 2080, ranging from more conservative to more aggressive, and representing the potential variation within which range shifts can be expected to occur. Future projections showed range shifts into regions where MPX has not been recorded previously. Increased suitability for MPX was predicted in eastern Democratic Republic of Congo. Models developed here are useful for identifying areas where environmental conditions may become more suitable for human MPX; targeting candidate reservoir species for future screening efforts; and prioritizing regions for future MPX surveillance efforts.     

Evidence for nonintercalative complexes formed from the reversible binding of benzo[a]pyrene metabolites to closed-circular, single-stranded M13mp19 DNA

The fluorescence excitation spectrum of complexes formed from the reversible binding of the proximate carcinogen, trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene (BP78D) to closed-circular, single-stranded, viral M13mp19 DNA (SS M13 DNA) exhibits a red-shift of 5 nm compared to the spectrum of BP78D measured without DNA or with native, calf thymus DNA. In SS M13 DNA which is 0.10 mM in PO4-, the fluorescence intensity of BP78D is 2.3 times smaller than the intensity measured without DNA; however, the fluorescence lifetime (42.7 nsec) of BP78D with SS M13 DNA is 1.7-1.8 times larger than the lifetimes of BP78D measured without DNA or with calf thymus DNA. These results are consistent with the conclusion that, in addition to binding sites which cause fluorescence quenching, SS M13 DNA contains sites which permit formation of BP78D inclusion complexes that have weaker interactions with nucleotide bases than those occurring in intercalated complexes. The association constant (1.45 +/- 0.01 x 10(5) M-1) for the binding of BP78D to SS M13 DNA is more than 9.0 times larger than that for binding to calf thymus DNA. It is 7.1 times larger than that for the binding of the less genotoxic metabolite, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (BP45D) to SS M13 DNA. UV Photoelectron data and results from ab initio molecular orbital calculations suggest that a difference in polarizability contributes to the greater SS M13 DNA binding of BP78D compared to that of BP45D.     

Evidence for the involvement of the thromboxane synthase pathway in human natural cytotoxic cell activity

We and other investigators have recently shown that inhibitors of lipoxygenase reversibly inhibit natural cytotoxic (NC) or natural killer (NK) cell activity, whereas some inhibitors of cyclooxygenase enhance these functions. In addition, exogenous LTB4 augments NC and NK activity, whereas PGE2 depresses it. In the present studies, we sought to investigate the possible role of the TxA2 synthase pathway in NC function. Inhibition of this pathway by OKY-1581 or dazoxiben significantly inhibited NC activity against HSV-infected cells as well as NK function against K562 target cells. The inhibition was dose dependent, reversible, and not due to direct toxicity. NC activity was also significantly inhibited by the addition of PGE2 or PGI2 to the 4-hr assay, whereas addition of 6-keto-PGF1 alpha had no effect. Addition of PGH2, which could be converted to TxA2 or other PG, had no significant effect, but concomitant use of OKY-1581 produced a greater inhibition of NC function than by using OKY-1581 alone. U44069, a TxA2 analog, was inhibitory by itself and could not alter the inhibition caused by OKY-1581 or dazoxiben. In contrast, the TxA2 receptor blocker 13-APA significantly enhanced NC activity and even reversed the inhibitory effect of U44069 at equimolar (10(-7)M) concentrations. Taken together, these data suggest that most of the inhibitory effect of the TxA2 synthase inhibitors on NC and NK cell function derives from their ability to reorient cyclic endoperoxide metabolism toward more inhibitory compounds. In addition, TxA2 itself could exert a negative feedback on NC function through its receptor, as evidenced by the use of a TxA2 analog and a TxA2 blocker.     

Influences of 7-alkyl substitution on the reversible binding of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene to DNA

The effects of 7-alkyl substitution on the reversible intercalation of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BAD) to calf thymus DNA have been examined using time-resolved fluorescence spectroscopy. The results indicate that in 10(-3) M sodium cacodylate the binding constant of BAD is 1.8 x 10(3) M-1. 7-Ethyl substitution decreases the binding constant 1.6 times, while 7-methyl substitution increases the binding constant 1.7 times. UV Photoelectron data and results from ab initio molecular orbital calculations suggest that an increase in polarizability contributes to the increased binding accompanying methyl substitution. The decreased binding accompanying ethyl substitution arises from steric inhibition. The physical binding data correlates with the decrease in carcinogenic activity which occurs with 7-ethyl substitution of benz[a]anthracene metabolites.