Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado.

Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process.

Genome-wide association analysis of clinical vs. nonclinical origin provides insights into Saccharomyces cerevisiae pathogenesis.

Because domesticated Saccharomyces cerevisiae strains have been used to produce fermented food and beverages for centuries without apparent health implications, S. cerevisiae has always been considered a Generally Recognized As Safe (GRAS) microorganism. However, the number of reported mucosal and systemic S. cerevisiae infections in the human population has increased and fatal infections have occurred even in relatively healthy individuals. In order to gain insight into the pathogenesis of S. cerevisiae and improve our understanding of the emergence of fungal pathogens, we performed a population-based genome-wide environmental association analysis of clinical vs. nonclinical origin in S. cerevisiae. Using tiling array-based, high-density genotypes of 44 clinical and 44 nonclinical S. cerevisiae strains from diverse geographical origins and source substrates, we identified several genetic loci associated with clinical background in S. cerevisiae. Associated polymorphisms within the coding sequences of VRP1, KIC1, SBE22 and PDR5, and the 5' upstream region of YGR146C indicate the importance of pseudohyphal formation, robust cell wall maintenance and cellular detoxification for S. cerevisiae pathogenesis, and constitute good candidates for follow-up verification of virulence and virulence-related factors underlying the pathogenicity of S. cerevisiae.

17-year follow-up of the rough-blasted threaded Weill cup in uncemented total hip arthroplasty.

INTRODUCTION: For uncemented hip arthroplasty, various cup designs are available. The threaded Weill acetabular component (Weill cup; Zimmer, Winterthur, Switzerland) has been used for more than 20 years, with poor results of the smooth threaded design. Our study was intended to assess the 17-year outcome of the rough-blasted option of the threaded Weill cup. MATERIALS AND METHODS: Between 1987 and 1988, a series of 86 rough-blasted threaded Weill cups were implanted in combination with the CLS Spotorno stem (Zimmer Ltd, Germany) The patients' mean age at the time of surgery was 50 years (range 19-67 years). 67 out of 86 hips (78%) were available for a follow-up at a mean of 17 years (range 16-18 years). Radiographs were available from 55 out of 63 unrevised hips (87%) and analyzed for radiolucency and PE wear. RESULTS: Two out of 86 cups (3%) were revised due to aseptic loosening and another two cups (3%) were awaiting revision for the same reason. Ten patients (10 cups, 12%) were lost to follow-up, and nine patients with nine cups (11%) had deceased without radiographic signs of cup failure. Cup survival with "revision or awaiting revision" as endpoint was 86% (95% CI 75-92%). No deep infections occurred, and no polyethylene insert was exchanged. The Harris hip score was excellent in 37 out of 67 clinically examined hips (55%), good in 18 hips (26%), satisfactory in 5 hips (8%) and moderate or poor in 5 hips (8%) and 2 hips (3%), respectively. CONCLUSION: The rough-blasted threaded Weill cup provides a good long-term performance in cementless total hip arthroplasty. The results compare favourably to the smooth threaded cup design.

[Periprosthetic femoral bone reaction after total hip arthroplasty with preservation of the collum femoris : CT-assisted osteodensitometry 1 and 3 years postoperatively]. / Periprothetische femorale Knochenreaktion nach schenkelhalserhaltender Hüftendoprothetik : CT-gestützte Osteodensitometrie 1 und 3 Jahre postoperativ.

BACKGROUND: Short-stemmed cementless femoral components in total hip arthroplasty have been designed to preserve the proximal femoral bone stock by load transfer to the femoral metaphysis. An in vivo method of computed tomography-assisted (CT) osteodensitometry after total hip arthroplasty is presented which differentiates between cortical and cancellous bone density (BD) changes around uncemented femoral components. PATIENTS AND METHODS: Cortical and cancellous periprosthetic femoral BD (mg Ca HA/ml) was determined prospectively in 31 patients at day 10, 1 year and 3 years after total hip arthroplasty with preservation of the collum femoris (C.F.P.-stem, Link, Hamburg, Germany) using computed tomography-assisted osteodensitometry. Clinical results (Harris hip score) and plain x-rays were assessed in all cases. RESULTS: Progressive proximal cortical BD loss was observed between the 1 year (Ø -8%) and 3 year (Ø -22%) postoperative measurements. Distal to the trochanter minor no significant cortical BD changes were observed. Proximal cancellous BD decreased progressively between the 1 year (Ø -33%) and 3 year (Ø -45%) analyses. The Harris hip score improved from 45 points pre-operatively to 93 points at the 3 year follow-up. All x-rays showed signs of stable ingrowth. CONCLUSION: Periprosthetic CT osteodensitometry has the technical ability to discriminate between cortical and cancellous bone structures with respect to strain-adapted remodeling. Progressive proximal cortical and cancellous BD loss indicates that metaphyseal fixation cannot be achieved with the analyzed C.F.P. stem design. The lack of cortical BD loss below the trochanter minor suggests diaphyseal fixation of the implanted stem.

Fungus-bacterium associations are widespread in fungal cultures isolated from a semi-arid natural grassland in Germany.

We report on a study that aimed at establishing a large soil-fungal culture collection spanning a wide taxonomic diversity and systematically screening the collection for bacterial associations. Fungal cultures were isolated from soil samples obtained from a natural grassland in eastern Germany and bacterial associations were assessed by PCR-amplification and sequencing of bacterial 16S rRNA. In addition, intraspecies genetic diversities of a subset of the isolated species were estimated by double-digest restriction associated DNA sequencing. A total of 688 fungal cultures, representing at least 106 fungal species from 36 different families, were obtained and even though clonal isolates were identified in almost all fungal species subjected to ddRAD-seq, relatively high genetic diversities could be observed in some of the isolated species. A total of 69% of the fungal isolates in our collection were found to be associated with bacteria and the most commonly identified bacterial genera were Pelomonas, Enterobacter and Burkholderia. Our results indicate that bacterial associations commonly occur in soil fungi, even if antibiotics are being applied during the isolation process, and provide a basis for the use of our culture collection in ecological experiments that want to acknowledge the importance of intraspecies genetic diversity.

Characterization of cytoskeletal proteins in follicular structures of cows with cystic ovarian disease.

The distribution of intermediate filaments (vimentin, cytokeratins, desmin) and microfilaments (alpha-smooth muscle actin and muscle specific actin) was studied immunohistochemically in bovine ovaries, with and without cystic ovarian disease. The immunohistochemically stained area (IHCSA), was quantified by image analysis, to evaluate the expression of these cytoskeletal proteins in the follicular wall of healthy antral, atretic, and cystic follicles. The granulosa cell layer of cystic follicles and atretic follicles had a significantly larger IHCSA for vimentin than did healthy antral follicles. Cytokeratins reacted lightly in the granulosa cells of antral follicles of normal ovaries, whereas granulosa cells of atretic and cystic follicles showed significantly higher IHCSA values. Immunohistochemical localization of desmin, muscle specific actin, and alpha-smooth muscle actin was restricted to the theca externa. This study supports earlier suggestions that strongly positive reactions with vimentin and cytokeratin antibodies observed in the granulosa cells of cystic follicles are due to the reorganization that occurs in the follicle during the process of cystic development, and are associated with changes in the expression of cytoskeletal proteins that are essential to proper cellular functioning.

AFLP-based assessment of the effects of environmental heavy metal pollution on the genetic structure of pioneer populations of Suillus luteus.

• The effects of environmental heavy metal pollution on the genetic structure of pioneer populations of the ectomycorrhizal basidiomycete Suillus luteus were assessed. • Sporocarps were collected from nine different locations and characterized by amplified fragment length polymorphism (AFLP) markers. Six of the sampling sites were contaminated with heavy metals and were dominated by tolerant individuals. Considerable genetic diversity was found within geographic subpopulations, but no reduction of the genetic diversity of populations inhabiting contaminated soils was observed. Neither did significant clustering of subpopulations inhabiting contaminated soils occur. Overall, the genetic differentiation between subpopulations was low, but Bayesian inference indicated the presence of two genetically differentiated clusters of individuals, which may correspond to different intercompatibility groups in S. luteus. • Heavy metal contamination seems to have a limited influence on the genetic structure of populations of S. luteus. Loss of diversity may have been prevented by sexual reproduction and rapid evolution of the tolerance trait or initial genetic bottlenecks may have been reduced by admixture and recurrent migration from surrounding populations colonizing noncontaminated soils.

Peripheral nervous system damage in experimental chronic Chagas' disease.

Electromyographic and histopathologic studies were performed in Rockland mice chronically infected with CA-I Trypanosoma cruzi strain. At 4 months post-infection the emg failed to show spontaneous activity, but a diminished interference pattern was detected in half of the infected group, while mean motor unit potential amplitude and duration were increased, compared with controls. An active denervation was observed at 6 months which persisted up to 9 months, when motor unit potential showed a significantly lower mean activity and duration. At 12 months most of the infected mice developed a reduced interference pattern, polyphasic motor unit potential increase with higher duration and amplitude than controls. Histopathologic studies showed myositis with perivascular involvement as well as intramuscular neuritis, especially at 4 and 12 months. Atrophic and hypertrophic fibers were seen. Few amastigote nests were detected. Inflammatory neuropathy with the demyelinated fibers and scanty axonal degeneration were the most common features in all infected mice. Mild myelinated fiber loss was only evident after 12 months. Endoneural parasites were seen only in the perineural macrophagic cells. These findings suggest that the neurogenic mechanism involved in the pathogenesis of muscle damage in this experimental model of chronic Chagas' disease consistently has been overlooked. The features registered here suggest that T. cruzi-infected mice developed a bimodal muscle denervation with an early acute period at any time before month 4, followed by reinnervation with a subsequent new acute denervation period by month 6, followed in turn by a slow later reinnervation.

Effect of vector on infectivity of Trypanosoma cruzi.

A comparative study was carried out on the interaction between Triatoma infestans and bloodstream forms (BSF) of Trypanosoma cruzi isolates RA and UP, both lethal for mouse, and CA-I nonlethal for this host. Parasite duplication was readily detected in triatomes fed with CA-I, metacyclic (Mtc) differentiation reaching a maximum at an optimum ingestion level of 250 BSF/insect. Progress and differentiation proved much lower for RA, but reached intermediate values for the UP isolate. Assays for infectivity for each isolate after bug passage revealed a drop for the RA and UP, whereas for CA-I an increase was observed indicating that virulence of BSF and Mtc differs. Our results suggest that parasite selection by insect passage modulates infectivity of a given parasite population; however, virulence was independent of the absolute number of Mtc in the insect's feces.

Analgesic activity of cyclic imides: 1,8-naphthalimide and 1,4,5,8-naphthalenediimide derivatives.

In early studies, we have reported the synthesis and biological activities of several cyclic imides. The present study describes the analgesic activity of 1,8-naphthalimide and 1,4,5,8-naphthalenediimide derivatives in a standard murine model of analgesia. The pharmacological results show that all compounds studied, given intraperitoneally, produced significant inhibition of acetic acid-induced abdominal constrictions. At the ID50 (micromol/kg) level, these cyclic imide derivatives were about 40-270-fold more potent in this assay than aspirin and acetaminophen, two well-known and widely used analgesics. These results extend previous studies on the analgesic activity of cyclic imides.

Immunogenicity of Trypanosoma cruzi strains determined by neutralization test.

Trypomastigotes of the CA-I strain of Trypanosoma cruzi, which is practically non-lethal for mice, exhibited adhered antibodies when harvested 25-28 days pi and handled at 4 degrees C, whereas at 37 degrees C they became negative within 10-15 min. Mice injected with these tryp pre-incubated with anti-RA rabbit serum (shown to contained neutralizing antibodies against the homologous strain) developed a significantly lower parasitemia than controls while those pre-incubated with anti-CA-I serum showed only a delay in the parasitemia pattern. Neutralizing activity was undetectable in anti-CA-I rabbit and mouse sera when the test was performed under standard conditions against RA-tryp. On the other hand, neutralizing antibodies were demonstrated in few anti-CA-I mouse serum samples against RA strain when the test was performed increasing the sensitivity in order to reach no more than 75% lethality for controls. In these cases, a close relationship between "infective dose size-immune response achieved" was found, being only a narrow range of inocula able to trigger detectable neutralizing activity (10(4) CA-I tryp inoculum). Variations in the degree of neutralizing activity as a function of the infective doze size was also demonstrated for anti-RA mouse sera and, despite every antiserum prepared with this T. cruzi strain was reactive, the best values were proved for those prepared with inocula up to 10(2) tryp. Above optimal parasite dose size, both strains induced a drop of neutralizing activity.

[Trypanosoma cruzi: differences in the requirement for protein protectors presented by 2 Argentinian strains]. / Trypanosoma cruzi: diferencias en el requerimiento de protectores proteicos presentadas por dos cepas argentinas.

The protein protector requirements for CA-I and RA T. cruzi trypomastigotes were standardized. The CA-I trypomastigotes needed a supplement of 5% serum or 5% bovine albumin in the washing and/or resuspending solutions to preserve their viability. For RA trypomastigotes, requirements were in the order of 1%. Trypomastigotes viability was determined by motility at the fresh microscopic observation and by mouse inoculation. This is a further demonstration of the particular behaviour reported for the CA-I bloodstream forms compared with those of RA.

Does retirement education teach people to save pension distributions?

As defined contribution pension plans have become increasingly common over the past two decades, so have lump sum distributions from those plans. Employees who elect such a distribution take the balance of their pension account with them when they leave a job. They can then choose to maintain the funds in accounts designated for retirement, invest them in other saving vehicles, or spend them. If spent pension distributions are not replaced by other savings, however, the future elderly are unlikely to be able to maintain a desirable standard of living. With employee-funded pensions expected to play an increasingly important role in financing Americans' retirement, saving these funds in essential. This article is the first to examine the relationship between retirement education--specifically, meetings sponsored by employers or by public and private institutions--and the saving of lump sum distributions. Two definitions of saving are used: one that includes reinvestment only in tax-deferred saving vehicles, and a broader one that includes tax-deferred vehicles, general saving vehicles (stocks, bonds, savings accounts, and so on), and paying off debt. The analysis also evaluates the effects of retirement education on specific groups identified in previous research as being less likely to keep their pension distributions in tax-deferred accounts: namely, women, younger persons, and persons with less than a college education. The same groups tend to be less financially secure in retirement, making the effects of retirement education on them particularly relevant. With an econometric model using ordinary least squares and data from the 1992 Health and Retirement Study, the analysis finds that retirement education does not affect the overall likelihood that employees will save their distributions, whether in tax-deferred or non-tax-deferred vehicles. The picture is more complicated for subgroups of employees. Attending a retirement meeting is associated with an increased likelihood of saving among persons age 40 and under but a decreased probability of saving among college graduates and women. No effect was found for men, individuals over age 40, or persons who did not graduate from college. The finding that retirement education increases the likelihood of younger persons' saving a distribution is reassuring, for these workers are America's future retirees. However, the finding that attending a meeting does not increase saving among some of the most financially vulnerable groups is a matter of concern to policymakers. Further study of the long-term effects of spending pension distributions is needed.

Gene expression profiling of a Zn-tolerant and a Zn-sensitive Suillus luteus isolate exposed to increased external zinc concentrations.

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis.

Estrogen receptors alpha and beta and progesterone receptors in normal bovine ovarian follicles and cystic ovarian disease.

The purpose of this study was to determine by immunohistochemistry the expression of estrogen and progesterone receptors in ovarian follicular structures from cows with cystic ovarian disease (COD) and to compare these with normal ovarian structures. Secondary, tertiary, atretic, and cystic follicles were evaluated. The follicular cysts of animals with COD presented a significantly higher expression of estrogen receptor alpha in all follicular layers than secondary, tertiary, and atretic follicles in both groups (P < .05). The intensity of estrogen receptor beta in the granulosa cell layer was stronger in tertiary than in secondary and atretic follicles in normal animals (P < .05) and in growing and cystic follicles in animals with COD (P < .05). Theca cells were scarcely stained in the 2 groups. Growing follicles and cysts from COD animals were less stained than tertiary follicles from normal animals (P < .05). Differences did not exist between the 2 groups with regard to the progesterone receptor. Ovaries of animals with COD exhibited altered estrogen receptors expression compared with that in normal animals.