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Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism.

Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 x 10(5) transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10(-6) to 19 x 10(-6) transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent.

Culture Media↗

Antibiotic-resistant cell-detaching Escherichia coli strains from Nigerian children.

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding alpha-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.

Bacterial Typing Techniques↗

Comparison of Borrelia burgdorferi sensu lato strains isolated from specimens obtained simultaneously from two different sites of infection in individual patients.

The aim of the present study was to analyze and compare Borrelia strains isolated from two different specimens obtained simultaneously from individual patients with Lyme borreliosis. Fifty such patients and 50 corresponding pairs of Borrelia isolates (100 low-propagated strains) were subjected to genotypic and phenotypic analysis, including pulsed-field gel electrophoresis for species identification and plasmid profile determination and protein profile electrophoresis for the assessment of the presence and molecular masses of separated proteins. The strains were isolated from two distinct skin lesions (12 patients), skin and blood (28 patients), skin and cerebrospinal fluid (8 patients), and blood and cerebrospinal fluid (2 patients). Out of 100 isolates, 63 were typed as B. afzelii and 37 as B. garinii. From each individual specimen only a single Borrelia species was cultured. Comparison of 50 Borrelia strain pairs isolated from two different specimens of an individual patient revealed that 12/50 (24%) patients were simultaneously infected with two different Borrelia strains; in 3/50 (6%) patients strains differed at the species level, in 4 out of the remaining 47 (9%) patients a strain difference in plasmid profile was established, while 5 out of the remaining 43 (11%) patient strain pairs differed in regard to the protein profiles of the two concurrently isolated strains. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor different B. burgdorferi sensu lato strains.

Borrelia↗

Embryonic stem cells: a perfect marriage between gene regulation and regenerative medicine.

The mechanism of mammalian gene regulation is highly complex, involving multiple layers of feedback control loops and dynamic chromatin remodeling. The current approach used to dissect the genetic circuitry of mammalian gene regulation utilizes somatic cells and protein fusion as a means to modulate protein interactions. This approach has several limitations that include (i) genome inaccessibility, (ii) high background interferences and, (iii) limited cellular phenotypes. Previously, the two broad fields of research "control of gene expression" and "stem cell biology" had been pursued separately by cell biologists; this review outlines evidence suggesting that integration of these two fields would provide a comprehensive platform for interdisciplinary research seeking to address mechanistic questions concerning gene regulation-that could have enormous implication for the development of therapeutic applications.

Animals↗

Phylogenetic and molecular evidence for allochronic speciation in gall-forming aphids (Pemphigus).

Sympatric populations can diverge when variation in phenology or life cycle causes them to mate at distinctly different times. We report patterns consistent with this process (allochronic speciation) in North American gall-forming aphids, in the absence of a host or habitat shift. Pemphigus populi-transversus Riley and P. obesinymphae Aoki form a monophyletic clade within the North American Pemphigus group. They are sympatric on the eastern cottonwood, Populus deltoides (Salicaceae), but have distinctly different life cycles, with sexual stages offset by approximately six months. Field evidence indicates that intermediate phenotypes do not commonly occur, and mitochondrial and bacterial endosymbiont DNA sequences show no maternal gene flow between the two species. Because a genetically distinct population of P. obesinymphae occurs in the southwestern United States on Populus fremontii, we consider the possibility of an initial allopatric phase in the divergence. We discuss the likely origins of the host use patterns in P. obesinymphae, and the larger sequence of evolutionary changes that likely led to the sympatric divergence of P. populi-transversus and P. obesinymphae. A plausible interpretation at this stage of investigation is that a shift in timing of the life cycle in an ancestral population, correlated with an underlying phenological complexity in its host plant, spurred divergence between the incipient species.

Animals↗

[Recent findings on the genetics of gastro-intestinal nematode resistance in ruminants].

The control of helminthiases in ruminants raised in open pasture has been mainly undertaken by using prophylactic measures in the environment, but these are often inadequate due to incorrect application. With the appearance of anthelmintics, the strategy for controlling these parasitoses, passed to pharmacological treatments which became effective in reducing their impact. However, the frequent and incorrect utilisation of these molecules resulted in resistance to anthelmintics and the presence of chemical residues in animal products for human consumption. Anthelmintic resistance is widespread throughout the world, heterogeneous and probably underestimated. This has encouraged the introduction of homeopathic agents and products derived from plants whose effectiveness has not been scientifically assessed. It is well known that it is possible to detect differences in resistance to the most important parasites between breeds. In Europe, it has been reported that some ovine autochthonous breeds, Scottish Blackface and Lacaune, showed higher resistance. The implementation of breeding strategies aimed at obtaining animals with naturally low susceptibility to nematode infestations could therefore play an increasingly important role. Standard animal breeding techniques have been largely successful in improving the performance of domestic animals in the last century. Standard quantitative selection requires field data on: i) individual phenotype performance; ii) expected covariance among animals due to blood relationship between them. The whole process of predicting the breeding value of animals in order to select subsequently the genetically superior parents of the next generation is entirely based on sophisticated computations (BLUP-animal model). In sheep, the main objective is always selecting for milk yield and sometimes, in addition, milk composition. However, due to the evolution of the EU agricultural policy and consumer demand in terms of healthy and organic food, more attention is now being given to traits related to health (resistance to EST, mastitis or parasitic diseases). Some studies conducted in New Zealand and Australia showed that nematode resistance is genetically controlled with high heritabilities and quite low genetic correlations with production traits. In this sense, some studies showed that it is possible to decrease the number of parasites in the framework of a traditional breeding programme. However, in most situations, this trait is not extensively recorded due to the high cost of individual recording. Therefore, it would be useful to implement breeding strategies based on the knowledge of the genes involved in this trait expression. Traditionally, two approaches are available to locate a gene: i) genome scan; ii) candidate gene approach. The candidate gene approach attempts to link general resistance to some particular genes. To date, genetic resistance against parasites is considered to be linked with the MHC and IgE genes. Furthermore, several gene detection studies based on the genome scan approach for this trait are currently being carried out on both crossed experimental populations (fat x lean Blackface lines and Sarda x Lacaune) and pure breeds (Churra). The preliminary results seem promising as to the use of marker assisted or genotype assisted selection for this trait, which is difficult and expensive to measure on a population scale.

Animals↗

[Functional plasticity of mammalian skeletal muscles under microgravity].

In the retrospective analysis of own experimental data and the literary overview of the slow-to-fast transformation of skeletal muscles (essentially postural and locomotor) in animals due to microgravity the authors scrutinize the muscle functional plasticity given heterodromous changes in the external mechanic field. Adaptation of the myofiber phenotype is ensured by polymorphism of muscle isoproteins and lability of the system providing energy for muscle contraction.

Adaptation, Physiological↗

[Diagnosis of uveitis with laboratory tests and specialized examinations].

The authors evaluated the importance of a large scale of laboratory examinations and specialized examinations in a group of 174 patients with endogenous uveitis examined at the uveological ambulance of the First Ophthalmological Clinic of the First Faculty, Charles University Prague. Laboratory examinations which are part of screening examinations are of little value for the diagnosis of uveitis. Alone they are unable to assess its type or prognosis. They cannot be recommended for routine work in the field. Only the incidence of phenotype HLA B27 in patients with anterior uveitis is, as compared with the healthy population, statistically significant (p < 0.005). Aimed collaboration with specialists from other disciplines is more important. The latter can on the basis of aimed examinations confirm or rule out systemic disease of a patient with uveitis suspected by the ophthalmologist.

Female↗

Clinical expression of X-linked retinitis pigmentosa in a Swedish family with the RP2 genotype.

PURPOSE: To examine the clinical phenotype with emphasis on electroretinograms and visual fields in a Swedish family with X-linked retinitis pigmentosa (XLRP) type 2 (RP2), and compare it with Swedish XLRP families with the RP3 genotype. METHODS: Three affected brothers and their carrier mother were examined clinically and with kinetic perimetry, dark adaptation thresholds, and full-field electroretinograms. The genotype was determined by haplotype analysis using polymorphic markers spanning the XLRP loci at the short arm of the X chromosome. RESULTS: The phenotype was consistent in the three affected males. The first subjective symptom was night blindness and the visual disability was more pronounced with increasing age. Affected individuals had a slight decrease in visual acuity and were emmetropic. They demonstrated a pathologically elevated final rod threshold. The visual fields were constricted in a somewhat atypical pattern. The three patients had an early presenting atypical cataract with multiple opacities. The fundus appearance was typical for RP with narrowing of retinal vessels and bone spicule pigmentations. The rod electroretinograms were extinguished in both eyes of the patients. The combined rod-cone responses as well as the isolated cone responses were severely reduced in amplitude; however, atypically for RP, the implicit time for the isolated cone responses was normal. The carrier female demonstrated normal ophthalmological findings, with the exception of two minimal pigmentations in the lower quadrants of the left eye. Haplotype analysis demonstrated that the disease in this family segregates with the RP2 locus. CONCLUSION: The phenotype of the studied RP2 family is associated with early onset of night blindness, emmetropia, a slight decrease in visual acuity, constriction of visual fields, and atypical cataract formation. Electroretinograms demonstrate severe rod dysfunction and surprisingly normal cone response implicit times which may indicate a milder disease progression. These findings are different from earlier descriptions of the RP2 and RP3 phenotypes.

Adolescent↗

High gene flow levels lead to gamete wastage in a desert spider system.

Field censuses, breeding experiments, and a quantitative model are used to obtain insight into the extent and consequences of genetic mixing between locally adapted populations of a desert spider. Typically, 9% of the matings of desert riparian spiders (non-aggressive phenotype) in native habitat involve an arid-land partner (aggressive phenotype). Mating was found to be random with respect to behavioral phenotype, but linearly related to both the rate of immigration and survival of immigrants from surrounding arid habitats in the riparian area. Genetic mixing between riparian and arid-land spiders produces offspring that exhibit lower rates of survival in riparian habitat. Two extreme behavioral phenotypes were also observed in the field studies: approximately 5% of the female spiders attacked all males they encountered while another 22% ran from all potential mates. Punnett square analyses of the potential genotypes produced by introgression between arid- and riparian-adapted spiders indicate that these extreme phenotypes appear in F2 generation hybrids and backcrosses. Because there is a costly wastage of gametes in the case of mixed phenotype mating, model results indicate that within three generations of the cessation of gene flow, the riparian population would be free of mixed genotypes and moving towards genetic differentiation.

Alleles↗

Biodiversity of the bacterial flora on the surface of a smear cheese.

The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.

Actinomycetales↗

Catalase deficiency reduces survival and pleiotropically affects agronomic performance in field-grown barley progeny.

Field-grown plants of the catalase-deficient mutant RPr79/4 show necrotic lesions in leaves and preferentially die. Initially, necrotic lesions exhibited by RPr79/4 were used to indirectly assess the role of distinct levels of catalase on the survival and agronomic performance of field-grown barley progeny. The segregation of three control traits was also analyzed to eliminate the influence of any obvious meiotic disturbance in case a reduction of plant survival was observed. The RPr79/4 necrotic phenotype had recessive expression in field-grown F1 plants. F2 progeny studies performed in the greenhouse revealed that the inheritance of necrotic lesions was monofactorial, and that the control traits segregated as expected. Progeny test analyses of field-grown F2 plants demonstrated that necrotic homozygous plants died preferentially. While the few surviving necrotic homozygous families were catalase-deficient, healthy homozygous families had normal levels of catalase. Progeny test analyses of the control traits confirmed the inheritance calculated in F2. Taken together, these findings indicate that abnormal segregation of necrotic lesions cannot be attributed to any obvious abnormal meiotic behavior but to the incapacity of catalase-deficient plants to overcome field stress conditions. Thus, catalase deficiency in barley reduced survival and pleiotropically affected the agronomic performance by diminishing seed weight and yield.

Journal Article↗

Stimulation of skeletal-derived cell cultures by different electric field intensities is cell-specific.

Pulsed electric stimulation, coupled capacitively to different cell cultures of skeletal origin, caused immediate changes in the cellular levels of cyclic AMP and a later enhanced DNA synthesis. Changes both in cyclic AMP level and DNA synthesis were correlated with the strength of the applied electric field. Cultures of calvaria bone cells which contain mainly two cell types, parathyroid hormone responsive cells (osteoblast-like) and prostaglandin E2 responsive cells (fibroblast-like), respond to both low (13 V/cm) and to high (54 V/cm) electric field strength, with no response at intermediate (24 V/cm) field strength. Rat epiphyseal cartilage responded like bone cells both to low and high field intensities, while rat condylar cartilage responded only to the intermediate field strength. Moreover, subcultures of calvaria bone cells, which lost their osteoblastic phenotype expression during subculturing, were responsive only to low field strength. On the other hand, osteoblast-enriched cultures, derived from calvaria bone grown in low calcium, were responsive only to the high field strength. These findings suggest that the response to various electric field intensities is cell-specific and might be used as an additional parameter to characterize cell types. Our study points to the possibility that when exposing a whole organ to an electrical stimulation it is possible to affect specifically only one cell population out of the many cell types existing in the organ.

Animals↗

Approaches to the genetics of cardiovascular disease through genetic field work.

Successful molecular genetic studies of complex disease require exact, careful phenotypization, which is more difficult than that performed for monogenic diseases. We have developed a family-oriented field working approach, which relies on index patients, their primary care physicians, and a minimum number of field working staff. The patients are responsible for recruiting their family members. Packets containing an explanatory pamphlet, an informed consent statement, a questionnaire, and blood cuvettes are provided. Data are transferred from questionnaire and from the laboratory into a computer program that facilitates construction of the family tree. We have applied this genetic field working approach primarily to patients with lipid disorders. Coupling results from genetic field working with modern DNA diagnostic tests such as the oligonucleotide ligation assay, has enabled us to effectively identify patients with familial hypercholesterolemia in the German population. We are now extending genetic field working to hypertension. Hypertension is much more difficult to study, because the phenotype is more difficult to discern and document. Both complex diseases have the disadvantage that the parents of the index patients are likely to already be dead. Nevertheless, we concentrate on the recruitment of large pedigrees, sibling pairs with parents whenever possible, and trios consisting of index patient and both parents or index patient, parent and sibling. With these constellations we can conduct association studies, linkage analysis, and novel combinations of both approaches.

Automation↗

Engineering of vaginal tissue in vivo.

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.

Actins↗

Persistent bacteremia due to methicillin-resistant Staphylococcus aureus infection is associated with agr dysfunction and low-level in vitro resistance to thrombin-induced platelet microbicidal protein.

BACKGROUND: The causes of persistent bacteremia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood. This investigation examined potential associations between PB with key clinical features and several in vitro bacterial genotypic and phenotypic characteristics, in isolates from 1 institution. METHODS: Pulsed-field gel electrophoresis (PFGE) relatedness, thrombin-induced platelet microbicidal protein (tPMP)-susceptibility phenotype, accessory gene regulator (agr) genotype and functionality (via delta-lysin production), and autolysis phenotypes were assessed in MRSA isolates from the bloodstream of 21 prospectively identified patients with PB (blood cultures positive after > or =7 days of therapy) and of 18 patients with resolving bacteremia (RB) (sterile blood cultures within the first 2-4 days of therapy) due to MRSA. RESULTS: The 2 groups had comparable baseline characteristics but differed in their clinical courses (e.g., endocarditis was more frequent in patients with PB than in those with RB [43% vs. 0%, respectively; P=.0016]); isolates from patients with PB exhibited higher rates of (1) survival in vitro after exposure to tPMP (22.4+/-14.8% vs. 11.6+/-6.5%, respectively; P=.005); (2) defective delta-lysin production (71.4% vs. 38.9%, respectively; P=.057); (3) non-agr genotype II profile (100% vs. 77.8%, respectively; P=.037); and (4) overrepresentation of a specific PFGE genotype (85.7% vs. 44.4%, respectively; P=.015). CONCLUSIONS: Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome.

Anti-Bacterial Agents↗

Mutations in wheat showing altered field resistance to yellow and brown rust.

Intensive screening of a small population of mutagenised wheat lines revealed a large number of lines with altered resistance to both yellow and brown rust. The parental cultivar Guardian has an intermediate level of adult plant resistance to this disease; mutants were therefore isolated that showed either enhanced resistance or enhanced susceptibility to yellow rust. Seven lines were identified that gave an altered yellow rust disease phenotype as adult plants under both field and greenhouse conditions. Simultaneous field testing for brown rust infection identified two of these lines as having increased resistance to brown rust.

Fungi↗

Dynamics of a novel pathogen in an avian host: Mycoplasmal conjunctivitis in house finches.

In early 1994, a novel strain of Mycoplasma gallisepticum (MG)--a poultry pathogen with a world-wide distribution--emerged in wild house finches and within 3 years had reached epidemic proportions across their eastern North American range. The ensuing epizootic resulted in a rapid decline of the host population coupled with considerable seasonal fluctuations in prevalence. To understand the dynamics of this disease system, a multi-disciplinary team composed of biologists, veterinarians, microbiologists and mathematical modelers set forth to determine factors driving and influenced by this host-pathogen system. On a broad geographic scale, volunteer observers ("citizen scientists") collected and reported data used for calculating both host abundance and disease prevalence. The scale at which this monitoring initiative was conducted is unprecedented and it has been an invaluable source of data for researchers at the Cornell Laboratory of Ornithology to track the spread and magnitude of disease both spatially and temporally. At a finer scale, localized and intensive field studies provided data used to quantify the effects of disease on host demographic parameters via capture-mark-recapture modeling, effects of host behavior on disease and vice-versa, and the biological and genetic profiles of birds with known phenotypic characteristics. To balance the field-based component of the study, experiments were conducted with finches held in captivity to describe and quantify the effects of experimental infections on hosts in both individual and social settings. The confluence of these various elements of the investigation provided the foundation for construction of a general compartmentalized epidemiological model of the dynamics of the house finch-MG system. This paper serves several purposes including (i) a basic review of the pathogen, host, and epidemic cycle; (ii) an explanation of our research strategy; (iii) a basic review of results from the diverse multi-disciplinary approaches employed; and (iv) pertinent questions relevant to this and other wildlife disease studies that require further investigation.

Animals↗