Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice

Lymphocyte recruitment to intestinal tissues depends on β(7) integrins. In this study, we studied disease severity and lymphocyte recruitment into the small intestine in SAMP1/YitFc mice, which develop chronic ileitis with similarity to human Crohn's disease. To assess the role of β(7) integrins in chronic ileitis, we generated SAMP1/YitFc lacking β(7) integrins (SAMP1/YitFc Itgb7(-/-)) using a congenic strain developed via marker-assisted selection. We analyzed ileal inflammation in SAMP1/YitFc and SAMP1/YitFc Itgb7(-/-) mice by histopathology and the distribution of T and B lymphocytes in the mesenteric lymph nodes (MLNs) by flow cytometry. Short-term (18 h) adoptive transfer experiments were used to study the in vivo homing capacity of T and B lymphocytes. In both young (<20 wk) and old (20-50 wk) SAMP1/YitFc Itgb7(-/-) mice, ileitis was reduced by 30-50% compared with SAMP1/YitFc mice. SAMP1/YitFc Itgb7(-/-) mice showed a dramatic 67% reduction in the size of their MLNs, which was caused by a 85% reduction in lymphocyte numbers and reduced short-term B cell homing. Flow cytometric analysis revealed a highly significant decrease in the percentage of B cells in MLNs of SAMP1/YitFc Itgb7(-/-) mice. Cotransfer of SAMP1/YitFc MLN B cells but not SAMP1/YitFc Itgb7(-/-) MLN B cells along with CD4(+) T cells resulted in exacerbated ileitis severity in SCID mice. Our findings suggest that β(7) integrins play an essential role in spontaneous chronic ileitis in vivo by promoting homing of disease-exacerbating B cells to MLNs and other intestinal tissues.     

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.     

Identifying hybridizing taxa within the <Emphasis Type="Italic">Daphnia longispina</Emphasis> species complex: a comparison of genetic methods and phenotypic approaches

Daphnia galeata Sars, D. longispina O. F. Müller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.     

Spatial-pattern analysis in a territorial spider: evidence for multi-scale effects

Territorial animals maintain a certain distance to neighbouring conspecifics, presumably leading to a regular spatial pattern through social spacing. Nevertheless, most animal populations are assumed to show aggregation at certain distance ranges, reflecting the scale dependency of spatial patterns. Leucorchestris arenicola (Araneae: Sparassidae) is a burrow-living spider species that shows territorial behaviour against conspecifics. A multi-scale approach in spatial analysis revealed that territory owners had fewer neighbours than expected under spatial randomness at distances up to 6 m. Behavioural field experiments showed that territory owners were able to perceive and react to burrow constructing neighbours up to at least 4 m distance from their own burrow. At larger distances individuals were often more aggregated than expected under spatial randomness. Analysing adult and immature relationships showed attraction between different development stages at small distances and avoidance at larger distances. The analysis reveals diverse spatial patterns in a territorial and cannibalistic species, showing that both behaviour and environment affect pattern development at different distances. The study outlines the importance of multi-scale approaches for spatial analysis and the need for accompanying experiments to facilitate the interpretation of results.