Pollen competition as an asymmetric reproductive barrier between two closely related Silene species

Reproductive barriers are important determinants of gene flow between divergent populations or species. We studied pollen competition as a post‐mating reproductive barrier between Silene dioica and S. latifolia. Gene flow between these species is extensive, but early‐generation hybrids are rare. In an experiment with conspecific, heterospecific and 50 : 50 mixed pollinations in the two species, pollination treatments did not significantly affect seed set and seed weight. However, molecular determination of siring success after mixed pollinations showed that fewer than expected hybrids were produced in S. latifolia (18% hybrids) but not in S. dioica (51% hybrids). This constitutes an asymmetric post‐mating reproductive barrier and likely contributes to the rarity of early‐generation hybrids. Our study shows that pollen competition can be an effective barrier to hybridization between closely related species that likely acts in concert with other reproductive barriers.     

Replicative senescence as a barrier to human cancer

There is evidence that one critically short telomere may be recognized as DNA damage and, as a consequence, induce a p53/p21WAF- and p16INK4A-dependent G1 cell cycle checkpoint to cause senescence. Additionally, senescence via a p53- and p16(INK4A)-dependent mechanism can be induced by the over- or under-stimulation of certain signalling pathways that are involved in cancer. Central to this alternative senescence mechanism is the p14ARF protein, which connects oncogene activation, but not DNA damage, to p53 activation and senescence. We find that immortal keratinocytes almost invariably have dysfunctional p53 and p16 and have high levels of telomerase, but very often express a wild-type p14(ARF). Furthermore, when normal keratinocytes senesce they show a striking elevation of p16 protein, but not of p14(ARF) or its downstream targets p53 and p21(WAF). These results suggest that p16, rather than p14(ARF), is the more important gene in human keratinocyte senescence, but do not exclude a co-operative role for p14(ARF), perhaps in the induction of senescence by activated oncogenes in neoplasia. Regardless of mechanism, these results suggest that replicative senescence acts as a barrier to human cancer development.     

Origin and evolution of GBV-C/hepatitis G virus and relationships with ancient human migrations

The GB virus C/hepatitis G virus (GBV-C/HGV) is a newly identified human RNA virus, belonging to the Flaviviridae family. Persistent infection by GBV-C/HGV is common in humans, and genetically divergent isolates have been identified in different parts of the world. Due to the absence of a real pathogenic role of GBV-C/HGV in liver disease and its extremely low mutation rate, this virus is a potential marker to trace prehistoric links between human populations. In this study, origin and evolution of GBV-C/HGV were examined using a set of fully sequenced strains of worldwide origin. A first phylogenetic analysis, addressed to the short (255 nucleotides) NS5A overlapping coding region by the neighbor-joining method, suggested an ancient African origin of GBV-C/HGV. This notion was confirmed when the same analysis was applied to the genomic regions showing the lowest rate of synonymous substitutions, covering one-fourth (2184 nucleotides) of the total coding potential of the virus genome. By using a multivariate statistical method and extending the analysis to the complete coding region, fine details of the evolutionary history of GBV-C/HGV were further elucidated. By this approach, isolates from Southeast Asia appeared to be the most closely related to those of African origin, consistent with a major route of ancient human migrations from Africa to southeastern parts of the Asian continent. Received: 26 October 2000 / Accepted: 28 February 2001     

More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

Authentication of ancient human DNA results is an exceedingly difficult challenge due to the presence of modern contaminant DNA sequences. Nevertheless, the field of ancient human genetics generates huge scientific and public interest, and thus researchers are rarely discouraged by problems concerning the authenticity of such data. Although several methods have been developed to the purpose of authenticating ancient DNA (aDNA) results, while they are useful in faunal research, most of the methods have proven complicated to apply to ancient human DNA. Here, we investigate in detail the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing so, we also produce data that describes the efficiency of bleach incubation of bone powder and its relative detrimental effects on contaminant and authentic ancient DNA. We note that bleach treatment is significantly more detrimental to contaminant than to authentic aDNA in the bleached bone powder. Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination. This asymmetrical behavior of authentic and contaminant DNA can be used to identify authentic haplotypes in human aDNA studies.     

The human commensal yeast, Candida albicans, has an ancient origin

Candida albicans, the primary causative agent of candidiasis, is a ubiquitous member of the human flora and is capable of causing severe invasive disease. Despite its importance as a human pathogen, little is known concerning those factors creating and maintaining genetic diversity within the species and how extant strains reflect their evolutionary history. Based on nucleotide polymorphism frequencies, we estimated the time to a most recent common ancestor for the species to be about 3-16 million years, with variation due to molecular clock calibration. As C. albicans genotypes have broad geographic associations, this suggests that the origins of DNA sequence variation in extant populations coincided with early hominid evolution. This is consistent with an emerging view of a genetically complex organism that is able to survive under host immunity as an obligate commensal species.     

The Himalayas as a directional barrier to gene flow

High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal indicates that the Himalayas have been permeable to dispersals from the east. These genetic patterns suggest that this cordillera has been a biased bidirectional barrier.     

Photocrosslinkable gellan gum film as an anti-adhesion barrier

The purpose of this study was to develop a gellan gum-based film which could be photocrosslinked for medical applications. Gellan gum was grafted with cinnamate to yield the photo crosslinkable polymer (gellan gum-cin). This material had 14.7% of its d-galacturonic residues reacted with cinnamate groups and displayed maximum absorption at 254nm. Investigation of the photochemical properties showed that the crosslinking efficiency was 82% after 16min of UV irradiation. The anti-adhesion films prepared from gellan gum-cin polymers exhibited high gel contents (88±2%) and suitable mechanical properties. When implanted into rats, the gellan gum-cin film exhibited the most promising anti-adhesion potential in 2 out of 10 rats without forming any tissue adhesion. Furthermore, the gellan gum-cin film could effectively inhibit inflammation in rats based on the results of fluid leukocyte analyses. The gellan gum-cin film thus has potential in clinical applications.     

The use of otolith chemistry to characterize diadromous migrations

Chemical constituents in otoliths have become a valuable tool for fish ecologists seeking to reconstruct migratory patterns and life-history diversity in a wide range of species worldwide. This approach has proved particularly effective with fishes that move across substantial salinity gradients over the course of their life, including many diadromous species. Freshwater endmembers of several elemental and isotope ratios (e.g. Sr:Ca, Ba:Ca and (87)Sr:(86)Sr) are typically identifiably distinct from marine values, and often differ among freshwater tributaries at fine spatial scales. Because these chemical tags are generally incorporated in proportion to their ambient dissolved concentrations, they can be effective proxies for quantifying the presence, duration and frequency of movements between freshwater and marine habitats. The development of high precision probe-based analytical techniques, such as laser ablation inductively coupled plasma mass spectrometry (ICP-MS) and microbeam methods, has allowed researchers to glean increasingly detailed life-history profiles of these proxies across otoliths. Researchers are also combining multiple chemical proxies in an attempt to refine interpretations of habitat residence patterns. A thorough understanding of the spatial and temporal variation in water chemistry as well as environmental and physiological controls on incorporation of specific elements into otoliths is required for confident estimation of lifetime salinity experience. First some assumptions, methodological considerations and data processing options that are particularly relevant to diadromous otolith chemistry studies are discussed. Insights into diadromous migrations obtained from decades of otolith chemistry research, highlighting the increasingly recognized importance of contingent behaviour and partial migration are then discussed. Finally, areas for future research and the need to integrate otolith chemistry studies into comprehensive assessments of the effects of global environmental change are identified.     

Genomic clues to an ancient asexual scandal

Despite abandoning meiosis, the bdelloid rotifers have persisted for millions of years and given rise to hundreds of species. Several mechanisms - allelic variants with different functions, high effective population size, and resistance to radiation - may contribute to their success.     

Hypermutation of an ancient human retrovirus by APOBEC3G

Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome, but all are remnants of ancient retroviral infections and harbor inactivating mutations that render them replication defective. Nevertheless, as viral “fossils,” HERVs may provide insights into ancient retrovirus-host interactions and their evolution. Indeed, one endogenous retrovirus [HERV-K(HML-2)], which has replicated in humans for the past few million years but is now thought to be extinct, was recently reconstituted in a functional form, and infection assays based on it have been established. Here, we show that several human APOBEC3 proteins are intrinsically capable of mutating and inhibiting infection by HERV-K(HML-2) in cell culture. We also present striking evidence that two HERV-K(HML-2) proviruses that are fixed in the modern human genome (HERV-K60 and HERV-KI) were subjected to hypermutation by a cytidine deaminase. Inspection of the spectrum of mutations that are found in HERV-K proviruses in the human genome and HERV-K DNA generated during in vitro replication in the presence of each of the human APOBEC3 proteins unequivocally identifies APOBEC3G as the cytidine deaminase responsible for hypermutation of HERV-K60 and HERV-KI. This is a rare example of the antiretroviral effects of APOBEC3G in the setting of natural human infection, whose consequences have been fossilized in human DNA, and a striking example of inactivation of ancient retroviruses in humans through enzymatic cytidine deamination.     

The role of land bridges,ancient environments,and migrations in the assembly of the North American flora

Continental‐scale assembly of floras results from past and present in situ diversification in association with several external processes. Among these processes are the making and breaking of connections among landmasses. Connections among landmasses are constantly in flux as are the climates and landscapes along the connection corridors, so that these corridors, or land bridges, may either facilitate or restrict migration at a given time. Across land bridges, changing landscape‐level and organismal factors include the dispersal potential and vectors of propagules, competition, predation, and distributions altered by pathogens. Assembly of a flora is, therefore, the outcome of complex, interacting, temporally‐varying factors that render simplistic explanations unlikely. In the case of North America, the continent experienced ephemeral connections with adjacent regions via five land bridges over the last 100 Ma at different times and under different climates and specific landscape morphologies, including edaphic characteristics. Here, I emphasize the earliest of these connections, Beringia, which probably comprised an initially‐incomplete land bridge during the Cretaceous and Paleocene resulting from compression, fragmentation, and rotation of Asian‐North American sub‐blocks as North America began moving westward from the northern portion of the Mid‐Atlantic Ridge. During the same time, additional land was added to Beringia with accretion of terranes and the subduction of the northern edge of the Pacific Plate beneath the North American‐Asian Plates in the Eocene to form the Aleutian Islands. Other connections between North America and adjacent landmasses were the North Atlantic, the Antilles, Central America, and the Magellan land bridge.     

Phylogeographic evidence for the existence of an ancient biogeographic barrier: the Isthmus of Kra Seaway

Biogeographic boundaries are characterised by distinct faunal and floral assemblages restricted on either side, but patterns among groups of taxa often vary and may not be discrete. Historical biogeography as a consequence, while providing crucial insights into the relationship between biological diversity and earth history, has some limitations. Patterns of intraspecific molecular variation, however, may show unambiguous evidence for such historical divides, and can be used to test competing biogeographic hypotheses (often based on the dispersal-vicariance debate). Here, we utilise this method to test the hypothesis that a major biogeographic transition zone between the Sundaic and Indochinese biotas, located just north of the Isthmus of Kra in SE Asia, is the result of Neogene marine transgressions that breached the Isthmus in two locations for prolonged periods of time (>1 million year duration). Phylogeographic analyses of a freshwater decapod crustacean, the giant freshwater prawn Macrobrachium rosenbergii, strongly supports the historical existence of the more northerly postulated seaway. Results presented here highlight the power of utilising intraspecific molecular variation in testing biogeographical hypotheses.     

The retrieval of ancient human DNA sequences.

DNA was extracted from approximately 600-year-old human remains found at an archaeological site in the southwestern United States, and mtDNA fragments were amplified by PCR. When these fragments were sequenced directly, multiple sequences seemed to be present. From three representative individuals, DNA fragments of different lengths were quantified and short overlapping amplification products cloned. When amplifications started from <40 molecules, clones contained several different sequences. In contrast, when they were initiated by a few thousand molecules, unambiguous and reproducible results were achieved. These results show that more experimental work than is often applied is necessary to ensure that DNA sequences amplified from ancient human remains are authentic. In particular, quantitation of the numbers of amplifiable molecules is a useful tool to determine the role of contaminating contemporary molecules and PCR errors in amplifications from ancient DNA.     

Modeling the bifurcating flow in an asymmetric human lung airway

In a former paper, the inspiratory flow characteristics in a three-generation symmetric bifurcation airway have been numerically investigated using a control volume method to solve the fully three-dimensional laminar Navier-Stokes equations. The present paper extends the work to deal with asymmetric airway extracted from the 5th-11th branches of the model of Weibel (Morphometry of the Human Lung. New York Academic Press, Verlag, 1963) in order to more appropriately model human air passage. Computations are carried out in the Reynolds number range 200-1600, corresponding to mouth-air breathing rates ranging from 0.27 to 2.16l/s, representative for an averaged height man breathing from quiet to vigorous state. Particular attentions are paid to establishing relations between the Reynolds number and the overall flow characteristics, including flow patterns and pressure drop. The study shows that the ratios of airflow rate through the medial branches to that of their mother branches are the same, and this is also true for the ratios of airflow rate through the lateral branches. This partially explains why regular human breathing is not affected by airways of different sizes.     

The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

The disturbed blood-brain barrier in human glioblastoma

The aim of this article is to describe alterations of the blood-brain barrier (BBB) in gliomas. The main clinical problem of human gliomas is the edematous swelling and the dramatic increase of intracerebral pressure, also compromising healthy areas of the brain. According to our concept, one of the main reasons on the cellular level for these clinical problems is the loss or reduction of astroglial polarity. Astroglial polarity means the specific accumulation of potassium and water channels in the superficial and perivascular astroglial endfeet membranes. The most important water channel in the CNS is the astroglial water channel protein aquaporin-4 (AQP4) which is arranged in a morphologically spectacular way, the so-called orthogonal arrays of particles (OAPs) to be observed in freeze-fracture replicas. In brain tumors, but also under conditions of trauma or inflammation, these OAPs are redistributed to membrane domains apart from endfeet areas. Probably, this dislocation might be due to the degradation of the proteoglycan agrin by the matrix metalloproteinase 3 (MMP3). Agrin binds to the dystrophin-dystroglycan-complex (DDC), which in turn is connected to AQP4. As a consequence, agrin loss may lead to a redistribution of AQP4 and a compromised directionality of water transport out of the cell, finally to cytotoxic edema. This in turn is hypothesized to lead to a breakdown of the BBB characterized by disturbed tight junctions, and thus to the development of vasogenic edema. However, the mechanism how the loss of polarity is related to the disturbance of microvascular tight junctions is completely unknown so far.