Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Impaired gait may contribute to the increased rate of falls in cognitively impaired older people. We investigated whether gait under simple and dual task conditions could predict falls in this group. The study sample consisted of 64 community dwelling older people with mild to moderate cognitive impairment. Participants walked at their preferred speed under three conditions: (a) simple walking, (b) walking while carrying a glass of water and (c) walking while counting backwards from 30. Spatiotemporal gait parameters were measured using the GAITRite(®) mat. Falls were recorded prospectively for 12months with the assistance of carers. Twenty-two (35%) people fell two or more times in the 12month follow-up period. There was a significant main effect of gait condition and a significant main effect of faller status for mean value measures (velocity, stride length, double support time and stride width) and for variability measures (swing time variability and stride length variability). Examination of individual gait parameters indicated that the multiple fallers walked more slowly, had shorter stride length, spent longer time in double support, had a wider support width and showed more variability in stride length and swing time (p<0.05). There was no significant interaction between gait condition and faller status for any of the gait variables. In conclusion, dual task activities adversely affect gait in cognitively impaired older people. Multiple fallers performed worse in each gait condition but the addition of a functional or cognitive secondary task provided no added benefit in discriminating fallers from non-fallers with cognitive impairment.

The purpose of this study was to characterize, using the scanning electron microscope, the nature of root surfaces denuded by (1) root planing alone or (2) root planing plus citric acid treatment. Six teeth were extracted from three squirrel monkeys, and the coronal half of the root surface was planed to remove attached periodontal ligament fibers and cementum. Citric acid, pH 1, was topically applied to the denuded surfaces of 3 teeth. The surface of only root-planed specimens had an irregular surface which corresponded to a smear layer. Root surfaces that had received acid treatment after root planing exhibited funnel-shaped orifices of dentinal tubules, and intertubular zones with a fibrillar, mat-like morphology. The implications of the different root-surface characteristics are discussed.

Screening of aerobic culturable hydrocarbon (HC)-degrading microorganisms isolated from petroleum-polluted soils and cyanobacterial mats from Indonesia resulted in the collection of 33 distinct species. Eight bacteria, 21 fungi and 4 yeasts were identified to the specific level by molecular and phenotypic techniques. Bacterial strains belonged to the genera Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia and Mycobacterium. Four species are new and not yet described. Fungi belonged to Aspergillus, Penicillium, Fusarium, Amorphoteca, Neosartorya, Paecilomyces, Talaromyces and Graphium. Yeasts were Candida, Yarrowia and Pichia. All strains were cultivated axenically in synthetic liquid media with crude oil as sole carbon and energy source. After incubation, the detailed chemical composition of the residual oil was studied by gravimetric and gas-chromatographic techniques. Thirteen parameters for assessing the biodegradation potential were defined and computed for each strain. Maximum degradation was observed on the saturated HCs (n- and isoalkanes, isoprenoids), whereas aromatic HC degradation was lower and was related to the structural composition of the molecules. A principal components analysis (PCA) permitted grouping and classifying the strains as a function of their degradative capacities. It was shown that the most active strains produced polar metabolites which accumulated in the resins and asphaltene fractions. These fractions are highly resistant to microbial metabolism. No taxonomic trend could be defined between microbial phyla in terms of HC biodegradation activity.

The serine protease urinary plasminogen activator or urokinase (uPA), produced in abundance by many malignancies, plays a key role in tumor cell invasion and metastasis. uPA is localized within the malignant cell milieu via its cell surface receptor [uPA receptor (uPAR)], which is expressed by tumor and tumor-associated cells. In the present study, we have used a syngeneic model of rat breast cancer to directly evaluate the role of uPAR as a diagnostic and therapeutic target in metastatic breast cancer. A polyclonal antibody against the ligand-binding NH(2)-terminal domain of rat uPAR (ruPAR) was developed. This antibody recognizes ruPAR by both immunofluorescence and Western blot analysis. Recombinant ruPAR and ruPAR IgG displaced the binding of (125)I-labeled ruPAR IgG to rat prostate cancer cells (Dunning R3227 Mat Ly Lu) and breast cancer cells (Mat B-III) overexpressing ruPAR (Mat B-III-uPAR). ruPAR IgG also blocked the invasive capacity of these tumor cells in a dose-dependent manner. Mat B-III-uPAR cells were inoculated s.c. into the mammary fat pad of syngeneic female Fischer rats. On day 10 after tumor cell inoculation, animals were injected with (125)I-labeled preimmune or ruPAR IgG and then sacrificed at timed intervals. Maximum (125)I uptake was observed in primary tumors and in tissues commonly affected by tumor metastases (liver, spleen, lungs, and lymph nodes) at 12 h. Injection of (125)I-labeled preimmune or ruPAR IgG into normal non-tumor-bearing animals resulted in minimal basal levels of uPAR expression and established the specificity of the ruPAR IgG. Similar results were obtained by Northern blot and PCR analysis of mRNA isolated from tissues of normal and tumor-bearing animals. To evaluate the effectiveness of this antibody in tumor progression, ruPAR IgG (50-100 microg/day) was injected s.c. for 7 days (day 1-7) at the site of tumor cell inoculation (mammary fat pad), and animals were sacrificed at various time points for evaluation of tumor growth and metastases. Animals receiving ruPAR IgG showed a marked decrease in tumor growth and metastases as compared with control tumor-bearing animals receiving the same dose of preimmune rabbit IgG. Histological analysis of experimental primary tumors showed marked tumor necrosis that was due to increased tumor cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Together, these studies demonstrate the ability of anti-uPAR antibody to decrease tumor volume and detect the presence of microscopic occult tumor metastases in malignancies where uPA/uPAR play a key role in tumor progression.

Two genes (<em>MAT</em>1A and <em>MAT</em>2A) encode for methionine adenosyltransferase (<em>MAT</em>), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. <em>MAT</em>1A is expressed mostly in the liver, whereas <em>MAT</em>2A is widely distributed. We showed a switch from <em>MAT</em>1A to <em>MAT</em>2A expression in human hepatocellular carcinoma (HCC), which facilitates cancer cell growth. Using DNase I footprinting analysis, we previously identified a region in the <em>MAT</em>2A promoter protected from DNase I digestion in HCC. This region contains NF-kappa B and AP-1 elements, and the present study examined whether they regulate <em>MAT</em>2A promoter activity. We found nuclear binding of NF-kappa B and AP-1 to the <em>MAT</em>2A promoter increased in HCC. Tumor necrosis factor alpha (TNFalpha), which activates both NF-kappa B and AP-1, increased <em>MAT</em>2A expression in a dose- and time-dependent manner, binding of both NF-kappa B and AP-1 to the <em>MAT</em>2A promoter and <em>MAT</em>2A promoter activity, with the latter effect blocked by site-directed mutagenesis of the NF-kappa B and AP-1 binding sites. Blocking NF-kappa B with I kappa B super-repressor or AP-1 with dominant-negative c-Jun led to decreased basal <em>MAT</em>2A expression and prevented the TNF alpha-induced increase in <em>MAT</em>2A expression. Although blocking NF-kappa B had no influence on the ability of TNF alpha to increase AP-1 nuclear binding, blocking AP-1 with dominant-negative c-Jun prevented the TNF alpha-mediated increase in NF-kappa B binding. In conclusion, both NF-kappa B and AP-1 are required for basal <em>MAT</em>2A expression in HepG2 cells and mediate the increase in <em>MAT</em>2A expression in response to TNF alpha treatment. Increased trans-activation of these two sites also contributes to <em>MAT</em>2A up-regulation in HCC.

The goal of this project is to describe and understand the organismal composition, structure, and physiology of microbial ecosystems from hypersaline environments. One collection of such ecosystems occurs at North America's largest saltworks, the Exportadora de Sal, in Guerrero Negro, Baja California Sur. There, seawater flows through a series of evaporative basins with an increase in salinity until saturation is reached and halite crystallization begins. Several of these ponds are lined with thick (10 cm) microbial mats that have received some biological study. To determine the nature and extent of diversity of the microbial organisms that constitute these ecosystems, we are conducting a phylogenetic analysis using molecular approaches, based on cloning and sequencing of small subunit (SSU) rRNA genes (16S for Bacteria and Archaea, 18S for Eukarya). In addition, we report preliminary results on the microbial composition of a laminated community that occurs in a crystallized gypsum-halite matrix in near-saturated salt water. Exposure of the interior of these large (kilogram) wet, endoevaporite crystals reveals a multitude of colors: layers of yellow, green, pink, and purple microbiota. To date, analyses of these two environments indicate the ubiquitous dominance of uncultured organisms of phylogenetic kinds not generally thought to be associated with hypersaline environments.

OBJECTIVE

Alterations of the perinatal environment, which lead to increased prevalence of the metabolic syndrome in adulthood, program an upregulation of systemic and/or adipose tissue glucocorticoid metabolism (11 beta-hydroxysteroid dehydrogenase type 1 [11 beta-HSD-1]-induced corticosterone reactivation). We hypothesized that postnatal programming could modulate high-fat diet-induced adipose tissue dysregulation in adulthood.

METHODS

We compared the effects of chronic (since weaning) high- or low-fat diet in postnatally normofed (control) or overfed (programmed) rats.

RESULTS

Postnatal programming accentuated high-fat diet-induced overweight, insulin resistance, glucose intolerance, and decrease in circulating and epididymal adipose tissue adiponectin. Neither manipulation altered liver function. Postnatal programming or high-fat diet increased systemic corticosterone production, which was not further modified when both manipulations were associated. Postnatal programming suppressed high-fat diet-induced decrease in mesenteric adipose tissue (MAT) glucocorticoid sensitivity and triggered high-fat diet-induced increase in MAT glucocorticoid exposure, subsequent to enhanced MAT 11 beta-HSD-1 gene expression. MAT tumor necrosis factor (TNF)-alpha, TNF-receptor 1, interleukin (IL)-6, resistin, and plasminogen activator inhibitor-1 mRNAs were not changed by high-fat feeding in control rats and showed a large increase in programmed animals, with this effect further enhanced by high-fat diet for TNF-alpha and IL-6.

CONCLUSIONS

Our data show for the first time that postnatal manipulation programs high-fat diet-induced upregulation of MAT glucocorticoid exposure, sensitivity, and inflammatory status and therefore reveal the pivotal role of the environment during the perinatal period on the development of diet-induced adipose tissue dysregulation in adulthood. They also urge the need for clinical trials with specific 11 beta-HSD-1 inhibitors.

Despite the expanding use of Whole Body Vibration training among athletes, it is not known whether adding Whole Body Vibration training to the conventional training of sprint-trained athletes will improve speed-strength performance. Twenty experienced sprint-trained athletes (13 male symbol, 7 female symbol, 17-30 years old) were randomly assigned to a Whole Body Vibration group (n=10: 6 male symbol and 4 female symbol) or a Control group (n=10: 7 male symbol, 3 female symbol). During a 5-week experimental period all subjects continued their conventional training program, but the subjects of the Whole Body Vibration group additionally performed three times weekly a Whole Body Vibration training prior to their conventional training program. The Whole Body Vibration program consisted of unloaded static and dynamic leg exercises on a vibration platform (35-40 Hz, 1.7-2.5 mm, Power Plate). Pre and post isometric and dynamic (100 degrees/s) knee-extensor and -flexor strength and knee-extension velocity at fixed resistances were measured by means of a motor-driven dynamometer (Rev 9000, Technogym). Vertical jump performance was measured by means of a contact mat. Force-time characteristics of the start action were assessed using a load cell mounted on each starting block. Sprint running velocity was recorded by means of a laser system. Isometric and dynamic knee-extensor and knee-flexor strength were unaffected (p>0.05) in the Whole Body Vibration group and the Control group. As well, knee-extension velocity remained unchanged (p>0.05). The duration of the start action, the resulting start velocity, start acceleration, and sprint running velocity did not change (>0.05) in either group. In conclusion, this specific Whole Body Vibration protocol of 5 weeks had no surplus value upon the conventional training program to improve speed-strength performance in sprint-trained athletes.

The prevalence of Toxoplasma gondii in 84 free-range chickens (34 from the northern Pará state, and 50 from Rio Grande do Sul, the southern state) from Brazil, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 39 (46.4%) of 84 chickens with titers of 1:10 in one, 1:20 in two, 1:40 in four, 1:80 in seven, 1:160 in five, 1:320 in six, 1:640 in eight and>> or =1:1280 in six. Hearts and brains of 45 chickens with titers of 1:20 or less were pooled and fed to two T. gondii-free cats. Hearts and brains of 39 chickens with titers of 1:10 or higher were bioassayed in mice. Feces of cats were examined for oocysts. One cat fed tissues from 31 chickens with titers of less than 1:10 from Rio Grande do Sul shed T. gondii oocysts. T. gondii was isolated by bioassay in mice from 33 chickens with MAT titers of 1:20 or higher. All infected mice from 10 isolates died of toxoplasmosis. All 34 isolates (15 from Pará, 19 from Rio Grande do Sul) were genotyped using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2 and Apico. Eleven genotypes were revealed for Pará isolates and seven genotypes for Rio Grande do Sul. No genotype was shared between the two geographical locations. These data suggest that T. gondii isolates are highly diverse and genetically distinct between the two different regions in Brazil that are 3500 km apart.

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities.

The plantar pressure distributions for a large heterogeneous sample of feet (N = 107) were collected during barefoot standing using a capacitance mat. From these data, the function of the foot during standing was characterized. Peak pressures under the heel (139 kPa) were, on average, 2.6 times greater than forefoot pressures (53 kPa). Forefoot peak pressures were usually located under the second or third metatarsal heads. No significant relationship was found between body weight and the magnitude of peak pressure. The concepts of a transverse arch at the level of the metatarsal heads and a "tripod" theory of load distribution were not substantiated by this study. Load distribution analysis showed that the heel carried 60%, the midfoot 8%, and the forefoot 28% of the weightbearing load. The toes were only minimally involved in the weightbearing process. Examples of unusual distributions are shown; finally, a checklist is provided to aid the clinician in evaluating plantar pressure findings.

OBJECTIVE

The purpose of the study was to evaluate the demographics, etiology, and radiographic findings associated with hallux rigidus in patients treated surgically over a 19-year period in a single surgeon's practice.

METHODS

Patients treated for hallux rigidus by cheilectomy and metatarsophalangeal joint fusion were identified from 1981 to 1999. Patients who had diabetes, inflammatory arthritis, infectious arthritis, crystalline arthritis, multiple forefoot deformities, neuromuscular disorders, or had died were excluded. A chart review and evaluation of preoperative radiographs were completed on all eligible patients. All identified patients were invited for a follow-up examination that included standard and stress radiographs, range-of-motion testing, Harris mat study, gait analysis, first ray mobility measurement, and standardized questionnaire assessment.

RESULTS

One hundred ten of 114 (96.5%) patients with a diagnosis of hallux rigidus returned for the final evaluation. Eighty cheilectomy patients (93 feet) and 30 arthrodesis patients (34 feet) were evaluated. The mean age at onset in the current study was 43 years (13-70 years) and only six patients developed symptoms at an age of less than 20 years. Hallux rigidus was graded based on a five-grade clinical-radiographic system. The mean follow-up was 8.9 years. Ninety-five percent of patients with a positive family history had bilateral disease at the final follow-up. At the initial examination in the current study, 81% of patients had radiographic and clinical evidence of unilateral disease, but at the final follow-up 79% of patients had radiographic and clinical evidence of bilateral disease. Eleven percent of patients in the present series had pes planus based on either a positive Harris mat study and/or heel valgus. There was no evidence of an Achilles or gastrocnemius tendon contracture. Radiographic analysis found that the concurrent presentation of hallux valgus and hallux rigidus was not common. Ninety-three of 127 feet (73%) had a chevron or flat metatarsophalangeal joint. Thirty-five feet were noted to have mild or moderate metatarsus adductus. A long first metatarsal was no more common in patients with hallux rigidus than in the general population. The mean first ray elevatus was 5.5 mm and was well within acceptable limits of normal. The mean first ray mobility was 5 mm in arthrodesis patients and 5.8 mm in cheilectomy patients.

CONCLUSIONS

Hallux rigidus was not associated with elevatus, first ray hypermobility, a long first metatarsal, Achilles or gastrocnemius tendon tightness, abnormal foot posture, symptomatic hallux valgus, adolescent onset, shoewear, or occupation. Hallux rigidus was associated with hallux valgus interphalangeus, bilateral involvement in those with a familial history, unilateral involvement in those with a history of trauma, and female gender. Metatarsus adductus was more common in patients with hallux rigidus than in the general population but a clear correlation was not found. Additionally, a flat or chevron-shaped metatarsophalangeal joint was more common in hallux rigidus patients.

BACKGROUND

Meniscal allograft transplantation (MAT) was developed as a means of treating the symptoms of compartmental overload after meniscectomy. Despite more than 20 years of research in this field, many controversies still exist regarding meniscal transplantation.

OBJECTIVE

The aims of this study were to assess (1) the quality of the published studies on MAT; (2) the indications for this type of surgery; (3) the methods used for preservation, sizing, and fixation of the allograft; and (4) the clinical and radiographic outcomes of this procedure and its role in preventing osteoarthritis.

METHODS

Systematic review.

METHODS

Inclusion criteria for the articles were (1) English language, (2) peer-reviewed clinical studies with evidence levels 1 to 4, (3) reported clinical and/or radiological outcomes of MAT isolated or combined with other procedures, (4) minimum 12-month follow-up, (5) case series of at least 10 patients, and (6) a follow-up rate of at least 80% (no more than 20% of patients lost to follow-up).

RESULTS

A total of 55 studies matched the inclusion criteria (2 level 2, 7 level 3, and 46 level 4). The average Coleman methodology score was 49.73 ± 12.41 (range, 24-81). There was agreement in the literature regarding the indications for MAT: joint line pain and tenderness correlated with previous meniscectomy, young patients, without diffuse Outerbridge grade III and no grade IV cartilage damage, and a stable and well-aligned knee. Different graft types have been used: viable, fresh frozen, cryopreserved, and lyophilized. The most common method for graft sizing was plain radiography. Different fixation techniques have been described, with only a few studies comparing the clinical results of the different techniques and with no proven superiority of one method over the other. All the studies showed clinical improvement at last follow-up visit compared with preoperatively. The chondroprotective effect of MAT is still unclear.

CONCLUSIONS

Meniscal allograft transplantation seems to provide good clinical results at short-term and midterm follow-up, with improvement in knee function as well as acceptable complication and failure rates. Higher quality studies are necessary to better assess the potential chondroprotective effect of MAT and to identify differences in terms of outcomes between different surgical techniques.

A taxonomic study was performed on 26 strains isolated from microbial mats in Antarctic lakes of the Vestfold Hills and the McMurdo Dry Valleys. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the Rhodobacter group of the alpha-subclass of the Proteobacteria. Sequence similarity values for the strains with their nearest phylogenetic neighbours (Jannaschia, Octadecabacter and Ketogulonicigenium) ranged between 94.0 and 95.8%. DNA-DNA hybridizations and comparison of repetitive extragenic palindromic DNA-PCR (rep-PCR) fingerprinting patterns revealed that these strains are members of three distinct species. The isolates are Gram-negative, chemoheterotrophic, non-motile rods and their DNA G+C contents range from 59.4 to 66.4 mol%. Whole-cell fatty acid profiles are similar and the primary fatty acid in all the strains is 18 : 1 omega7c (74.1-87.7% of total). Genotypic results together with phenotypic characteristics allowed the differentiation of these species from related recognized species of the alpha-Proteobacteria and the strains are assigned to a new genus, Loktanella gen. nov., with three novel species: Loktanella salsilacus sp. nov. (type species), consisting of ten strains with LMG 21507T (=CIP 108322T) as type strain; Loktanella fryxellensis sp. nov., consisting of 12 strains with LMG 22007T (=CIP 108323T) as type strain; and Loktanella vestfoldensis sp. nov., consisting of four strains with LMG 22003T (=CIP 108321TT) as type strain.

Six phototrophic microbial mat communities from different geothermal springs (YNP) were studied using metagenome sequencing and geochemical analyses. The primary goals of this work were to determine differences in community composition of high-temperature phototrophic mats distributed across the Yellowstone geothermal ecosystem, and to identify metabolic attributes of predominant organisms present in these communities that may correlate with environmental attributes important in niche differentiation. Random shotgun metagenome sequences from six phototrophic communities (average ∼53 Mbp/site) were subjected to multiple taxonomic, phylogenetic, and functional analyses. All methods, including G + C content distribution, MEGAN analyses, and oligonucleotide frequency-based clustering, provided strong support for the dominant community members present in each site. Cyanobacteria were only observed in non-sulfidic sites; de novo assemblies were obtained for Synechococcus-like populations at Chocolate Pots (CP_7) and Fischerella-like populations at White Creek (WC_6). Chloroflexi-like sequences (esp. Roseiflexus and/or Chloroflexus spp.) were observed in all six samples and contained genes involved in bacteriochlorophyll biosynthesis and the 3-hydroxypropionate carbon fixation pathway. Other major sequence assemblies were obtained for a Chlorobiales population from CP_7 (proposed family Thermochlorobacteriaceae), and an anoxygenic, sulfur-oxidizing Thermochromatium-like (Gamma-proteobacteria) population from Bath Lake Vista Annex (BLVA_20). Additional sequence coverage is necessary to establish more complete assemblies of other novel bacteria in these sites (e.g., Bacteroidetes and Firmicutes); however, current assemblies suggested that several of these organisms play important roles in heterotrophic and fermentative metabolisms. Definitive linkages were established between several of the dominant phylotypes present in these habitats and important functional processes such as photosynthesis, carbon fixation, sulfur oxidation, and fermentation.

RNAi is a conserved mechanism in which small RNAs induce silencing of complementary targets. We have previously identified priRNAs, a class of Dicer-independent small RNAs in fission yeast. The mechanism by which Dicer-independent small RNAs are generated is not well understood in any species. Here we reconstitute the final steps of priRNA and siRNA biogenesis in vitro. We identify the 3'-5' exonuclease Triman and demonstrate that Argonaute, loaded with longer RNA precursors, recruits Triman to generate mature priRNAs and siRNAs. We show that priRNA and siRNA trimming is required for de novo assembly of heterochromatin at centromeric repeats and the mat locus and for maintenance of heterochromatin at developmental genes. Furthermore, in rrp6Δ cells RNAi targets diverse genes in a Triman-dependent way, indicating that the exosome protects the genome from spurious RNAi. Our results suggest that Argonaute association with RNA degradation products generates priRNAs and triggers RNAi in a process of transcriptome surveillance.

The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by "Bacilli" and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution.

In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast, for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed approximately 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.

The purpose of this study was to determine how diverse momentum conditions and anatomical orientation at contact influences mechanical loading and multijoint control of the reaction force during landings. Male collegiate gymnasts (n=6) performed competition style landings (n=3) of drop jumps, front saltos, and back saltos from a platform (0.72 m) onto landing mats (0.12 m). Kinematics (200 fps), reaction forces (800 Hz) and muscle activation patterns (surface EMG, 1600 Hz) of seven lower extremity muscles were collected simultaneously. Between-task differences in segment orientation relative to the reaction force contributed to significant between-task differences in knee and hip net joint moments (NJM) during the impact phase. During the stabilization phase, ankle, knee, and hip NJMs acted to control joint flexion. Between-task differences in muscle activation patterns indicated that gymnasts scaled biarticular muscle activation to accommodate for between-task differences in NJM after contact. Activation of muscles on both sides of the joint suggests that impedance like control was used to stabilize the joints and satisfy the mechanical demand imposed on the lower extremity. Between-subject differences in the set of muscles used to control total body center of mass (TBCM) trajectory and achieve lower extremity NJMs suggests that control of multijoint movements involving impact needs to incorporate mechanical objectives at both the total body and local level. The functional consequences of such a control structure may prove to be an asset to gymnasts, particularly when required to perform a variety of landing tasks under a variety of environmental constraints.

Collagens are the major proteinaceous constituents of cartilage. Three collagen types participate in the formation of striated fibrils of cartilage, collagens II, IX, and XI. Collagen II and XI belong to the subgroup of fibrillar collagens and are structurally closely related, differing mainly in their N-propeptides. Collagen IX has a very different structure but is nevertheless an essential constituent of the striated fibrils. Two other collagen types are also found in cartilage but form distinct structures. Collagen VI, found mainly in the periphery of the chondrocytes, forms beaded filaments. These filaments are probably formed by interaction of collagen VI with hyaluronan. Collagen X is expressed by hypertrophic chondrocytes. It has been shown to form in vitro hexagonal lattices and in vivo to be associated either with striated fibrils or with mats which may correspond to the lattices. The functional role of the collagen diversity in cartilage is discussed.

BACKGROUND

This study describes the ultrastructure of long-term denervated rat extensor digitorum longus and tibialis anterior muscles, with particular emphasis on understanding the cellular basis for the reduced restorative capacity of long-term denervated muscle.

METHODS

In 30 male WI/HicksCar rats, the right hindleg was denervated for periods of 1, 2, 4, 5.5, 6, 7, 12, 14, and 18 months before tissues were prepared for electron microscopy.

RESULTS

Atrophy of muscle fibers was prominent by the second month post-denervation. At this time, type II fibers showed greater atrophy than type I fibers. At further periods of denervation, atrophy of all fibers was seen; and with increasing times of denervation the muscle fibers became surrounded by dense mats of collagen fibers. Muscle spindles persisted for the duration of this study. At two and four months, satellite cells showed signs of activation, such as elongated cytoplasmic processes and an increased concentration of cytoplasmic organelles. As denervation progressed, activated satellite cells became more widely separated from their associated muscle fibers, and basal lamina material was deposited between the satellite cells and muscle fibers. Some satellite cells broke free from their muscle fibers, and others acted as bridges between two muscle fibers. Evidence was seen of both muscle fiber degeneration and the regeneration of new muscle fibers, often more than one regenerating fiber beneath a single basal lamina. Loose folds of basal lamina were often present around atrophic muscle fibers. As denervation progressed, the morphology of individual muscle fibers varied. Some contained well-ordered lattice arrays of myofilaments, whereas in others considerable sarcomeric disorganization was evident. Mitochondria became smaller and rounded; elements of the sarcoplasmic reticulum proliferated and became more disorganized; lipid droplets, glycogen deposits, and autophagic vesicles were all present in the cytoplasm of atrophic muscle fibers.

CONCLUSIONS

In addition to muscle fiber atrophy, long-term denervated muscles show evidence of myofiber and capillary death, as well as the deposition of massive amounts of interstitial collagen. These changes, all of which would appear to reduce the restorative capacity of the denervated muscle, take place concurrently with the morphological activation of satellite cells. The latter indicates that even in the denervated condition, restorative processes occur concurrently with regressive processes.

Evergreen broad-leaved tropical forests can have high rates of productivity and large accumulations of carbon in plant biomass and soils. They can therefore play an important role in the global carbon cycle, influencing atmospheric CO2 concentrations if climate warms. We applied meta-analyses to published data to evaluate the apparent effects of temperature on carbon fluxes and storages in mature, moist tropical evergreen forest ecosystems. Among forests, litter production, tree growth, and belowground carbon allocation all increased significantly with site mean annual temperature (MAT); total net primary productivity (NPP) increased by an estimated 0.2-0.7 Mg C x ha(-1) x yr(-1) x degrees C(-1). Temperature had no discernible effect on the turnover rate of aboveground forest biomass, which averaged 0.014 yr(-1) among sites. Consistent with these findings, forest biomass increased with site MAT at a rate of 5-13 Mg C x ha(-1) x degrees C(-1). Despite greater productivity in warmer forests, soil organic matter accumulations decreased with site MAT, with a slope of -8 Mg C x ha(-1) x degrees C(-1), indicating that decomposition rates of soil organic matter increased with MAT faster than did rates of NPP. Turnover rates of surface litter also increased with temperature among forests. We found no detectable effect of temperature on total carbon storage among moist-tropical evergreen forests, but rather a shift in ecosystem structure, from low-biomass forests with relatively large accumulations of detritus in cooler sites, to large-biomass forests with relatively smaller detrital stocks in warmer locations. These results imply that, in a warmer climate, conservation of forest biomass will be critical to the maintenance of carbon stocks in moist tropical forests.

Along with decarbamoylsaxitoxin and decarbamoylgonyautoxin-2 and -3, six new saxitoxin analogues were isolated from the freshwater mat-forming filamentous cyanobacterium Lyngbya wollei collected from Guntersville Reservoir on the Tennessee River in Alabama. Their structures were determined by electrospray ionization mass spectrometry and several NMR techniques. Five of the toxins contain an acetyl moiety attached to the side chain, which is the first report of these saxitoxin analogues. In three of the toxins a hydrated ketone at C-12 was reduced to alpha-alcohol. The presence of acetate in the side chain resulted in a sevenfold to 17-fold times decrease in mouse toxicity compared to their carbamoyl counterparts, while the reduction at C-12 resulted in a complete loss of mouse toxicity.