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In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.

The continuing increase in antibiotic-resistant microorganisms is driving the search for new antibiotic targets and improved antimicrobial agents. Ketolides are semisynthetic derivatives of macrolide antibiotics, which are effective against certain resistant organisms. Solithromycin (CEM-101) is a novel fluoroketolide with improved antimicrobial effectiveness. This compound binds to the large 50S subunit of the ribosome and inhibits protein biosynthesis. Like other ketolides, it should impair bacterial ribosomal subunit formation. This mechanism of action was examined in strains of Streptococcus pneumoniae , Staphylococcus aureus , and Haemophilus influenzae . The mean 50% inhibitory concentrations (IC 50 s) for solithromycin inhibition of cell viability, protein synthesis, and growth rate were 7.5, 40, and 125 ng/ml for Streptococcus pneumoniae , Staphylococcus aureus , and Haemophilus influenzae , respectively. The net formation of the 50S subunit was reduced in all three organisms, with IC 50 s similar to those given above. The rates of 50S subunit formation measured by a pulse-chase labeling procedure were reduced by 75% in cells growing at the IC 50 of solithromycin. Turnover of 23S rRNA was stimulated by solithromycin as well. Solithromycin was found to be a particularly effective antimicrobial agent, with IC 50 s comparable to those of telithromycin and significantly better than those of azithromycin and clarithromycin in these three microorganisms.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2-3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3(-/-)/Nrl(-/-) mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3(-/-)/Nrl(-/-) mice compared with Nrl(-/-) mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors.

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild-type (WT) D2 loop consists of approximately 21% α-helix, approximately 20% β-sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild-type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in α-helicity. However, the thermal stability of the mutants was unchanged compared to wild-type, and all the mutants were capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.

Oncostatin M (OSM) and leukemia inhibitory factor are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family. These cytokines play a crucial role in diverse biological events like inflammation, neuroprotection, hematopoiesis, metabolism, and development. The family is grouped together based on structural similarities and their ability to activate the transmembrane receptor glycoprotein 130 (gp130). The common structure among these cytokines defines the spacing and the orientation of binding sites for cell surface receptors. OSM is unique in this family as it can signal using heterodimers of gp130 with either leukemia inhibitory factor receptor (LIFR) (type I) or oncostatin M receptor (OSMR) (type II). We have identified a unique helical loop on OSM between its B and C helices that is not found on other IL-6 family cytokines. This loop is located near the "FXXK" motif in active site III, which is essential for OSM's binding to both LIFR and OSMR. In this study, we show that the BC loop does not play a role in OSM's unique ability to bind OSMR. Shortening of the loop enhanced OSM's interaction with OSMR and LIFR as shown by kinetic and equilibrium binding analysis, suggesting the loop may hinder receptor interactions. As a consequence of improved binding, these structurally modified OSMs exhibited enhanced biological activity, including suppressed proliferation of A375 melanoma cells.

Objective: To identify Chlamydia trachomatis antigens that can be used to differentially diagnose tubal factor infertility in comparison with previously reported heat shock protein 60. Design: In vitro study. Setting: Academic medical center. Patient(s): Infertile women with and without tubal pathology diagnosed laparoscopically. Intervention(s): None. Main Outcome Measure(s): Antibody responses to C. trachomatis in infertile women with or without tubal pathologies using a C. trachomatis genome-wide proteome array. Result(s): Comparison of the antibody profiles revealed 30 C. trachomatis antigens that were preferentially recognized in women with tubal factor infertility, with a detection sensitivity and specificity of 80.6% and 56.5%, respectively, 10 of which showed 100% specificity. A combination of CT443 and CT381 antigens yielded the highest detection sensitivity (67.7%) while maintaining 100% specificity. Conclusion(s): These findings have demonstrated that antibodies to CT443 and CT381, when used in combination, have higher sensitivity and specificity in predicting tubal factor infertility than other indicators for tubal factor infertility, such as heat shock protein 60 antibodies (35.5%, 100%) or hysterosalpingogram (65%, 83%). Using a panel of C. trachomatis antigens to serologically diagnose tubal factor infertility can save the patients from undertaking expensive and invasive procedures for determining tubal pathology and choosing treatment plans. (Fertil Steril� 2011;96:715–21. � 2011 by American Society for Reproductive Medicine.)

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransducton. Mutations in the channel subunits are associated with achromatopsia and progressive cone dystrophy in humans. More than 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them located in the carboxyl (C) terminus. This study investigates the defects of the two frequently occurring mutations, R377W and F488L, in the C-terminus of CNGA3. Ratiometric measurement of the intracellular Ca(2+) concentration and electrophysiological recordings showed the loss of functional activity of the mutant channels in an HEK293 heterologous expression system. Immunofluorescence labeling revealed an apparent cytosolic aggregation of the mutant channels compared to the wild type (WT). The R377W and F488L mutants, expressed and purified from Escherichia coli as glutathione S-transferase (GST) fused to the CNGA3 C-terminal domain, showed no negative effects on interactions with the channel subunits. Circular dichroism spectrum analyses were performed to examine the structural impact of the mutations. Although the R377W and F488L C-termini mutants retained stable, folded structures, the secondary structures of both mutants differed from the WT protein. Furthermore, the WT C-terminus exhibited a significant decrease in alpha-helical content in response to the channel ligands, while this allosteric transition was diminished in the two mutants. This is the first study showing the structural impact of the disease-causing mutations in the cone CNG channel subunit. The observed alterations in the local secondary structure and active conformational change may confer an adverse effect on the channel's activity and cellular processing.

SummaryIt was the purpose of the present study to validate administrative claims codes for idiopathic thrombotic thrombocytopenic purpura (TTP) in a commercially-insured US population. Patients with at least one medical claim with ICD-9 code 446.6X between 1/1/2001 and 5/31/2008 were identified in the HealthCore Integrated Research Database™ (HIRD). A chart abstraction form was developed to enable case determination for patients identified by the claims code. Two clinical experts, not involved in the design of the study, reviewed the abstracted medical record data and determined whether definite evidence supporting the diagnosis of TTP was present. The positive predictive value (PPV) of the claims coding algorithm for cases assessed by both reviewers was computed. The claims algorithm was further refined and the PPV of the refined algorithm was computed. One hundred eighty-nine abstracted charts were reviewed by two clinical experts; 86 were assessed to have definite evidence supporting the diagnosis of TTP (PPV 45.5% [86/189; 95% confidence interval (CI), 38.3–52.9%]). Refinement of the claims algorithm first included the use of plasma exchange treatment, resulting in 103 potential cases, of which 67 were assessed to have definite evidence supporting the diagnosis of TTP (PPV 65.0%; 95% CI, 55.0–74.2%). Further refinement of the claims algorithm ruled out alternative diagnoses that may mimic TTP; 34 were assessed to have definite evidence supporting the diagnosis of TTP (PPV 72.3% [34/47; 95% CI, 57.4–84.4%]).Our findings demonstrate the difficulty of confirming the diagnosis of rare disorders that lack definite diagnostic criteria, and indicate that more complex claims coding algorithms are necessary for identifying these disorders.

Several in vitro models that attempt to replicate the intraperitoneal environment have been developed to study the pathogenesis of endometriosis. The chicken chorioallantotic membrane has been used, but it has not been well characterized and may introduce some species specific variables. In vitro models using human tissues include amniotic membrane, human peritoneal explants, and cell culture monolayers. These models have been used to qualitatively, quantitatively, and temporally assess attachment of endometrial cells to peritoneal mesothelial and subsequent transmesothelial invasion. These models have also been used to assess the role of cytokines in the development of the early endometriotic lesion. Two- and three dimensional invasion chamber models have been utilized to assess endometrial cell interactions with peritoneal mesothelial cells and the extracellular matrix. Invasion models are also useful to evaluate novel therapeutic approaches. This review will focus on the above models to assist reproductive scientists interested in the pathogenesis of endometriosis.

Senior Reach program outcomes for older adults referred for care management and mental health services through a Gatekeeper model were examined in this study and compared with the Spokane Gatekeeper model. The two programs were compared for seniors served on service variables and outcome ratings for isolation, depression, and functioning. Approximately 41% of seniors served by both programs were referred by nontraditional sources: community gatekeepers. Findings indicate that individuals served by the Senior Reach program demonstrated significant improvement in reduction of isolators (such as social isolation), improved functioning, increased optimism about the future, increased positive activities with others, decreased emotional disturbance, and improvements on the Geriatric Depression Scale. Additional program comparisons and findings are discussed. Findings for the Senior Reach program demonstrate that the gatekeeper approach to training community partners is effective in finding at-risk seniors and meeting their needs, resulting in positive impacts on their lives. Older adults confront numerous challenges that can seriously impact their mental health. Issues such as retirement, death of a loved one, changes in health, relocation, and adjusting to other life changes are examples of the conditions and life circumstances that older adults experience (Raschko, 1991). Although there are services that could help seniors in those circumstances, many do not seek assistance (Florio, Jensen, Hendryx, Raschko, & Mathieson, 1998). Seniors may fear the stigma attached to asking for help (Raschko, 1991), systems may be confusing, services may not be coordinated, funding may be lacking, or services may be fragmented (Bartels et al., 2004). In 1978, the Spokane Mental Health, Elder Services Division decided on two strategies to overcome these barriers, one of which was to develop a community network of trained persons called "Gatekeepers" who could make referrals for elders they identified as at-risk. The Gatekeeper model (see Florio et al., 1996, for a complete description) was created to bring at-risk older adults to the attention of professionals who could provide outreach through a care management program. In 2006, Jefferson Center for Mental Health, The Mental Health Center Serving Boulder and Broomfield Counties, and the Seniors' Resource Center partnered to create a program modeled after the Spokane Gatekeeper program. Previous studies of the Gatekeeper model had identified the need for mental health services for these at-risk older adults and the need for more research on this model. The new program incorporated those components and was named Senior Reach. THE SENIOR REACH PROGRAM Like the Spokane Gatekeeper model, the mission of Senior Reach is to support the well-being and independence of seniors by educating the community on how to identify and refer isolated, at-risk older adults who may benefit from a model of care management. Additionally, Senior Reach addresses the need for mental health treatment and information and referral assistance. Central to the Senior Reach program was the objective to implement the Gatekeeper model with fidelity to the Spokane, Washington program that had sought to improve several key outcomes of seniors' lives. Further, replicating the outcomes of the Spokane program could benefit and inform the implementation of the Gatekeeper model in other similar areas. Two mental health centers and a senior center collaborated to develop and implement Senior Reach. Together, they represent a five-county ( Jefferson, Gilpin, Clear Creek, Boulder, and Broomfield), contiguous area all near the metropolitan area of Denver, Colorado, representing urban, suburban, and rural communities where 8%-20% of the older adults are estimated to have depressive symptoms (U.S. Surgeon General, 1999), and where approximately 53% of older adults identified with severe needs and poverty did not receive care (TriWest Group, 2003). …

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn(2+) ions. Stripping the full complement of bound Zn(2+) ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn(2+) core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn(2+) ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn(2+) ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn(2+) coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn(2+) serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.

The RAG1 and RAG2 proteins catalyze the site-specific DNA cleavage reactions in V(D)J recombination, the process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, to consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction at physiological conditions is masked by a high affinity non-sequence specific DNA-binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence specific DNA, resulting in a large differential in binding affinity for the RSS versus non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS, and that therefore association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.

Endometriosis is a common chronic disease that causes symptoms of pain and infertility. The pain syndrome can be quite incapacitating. The pain symptoms usually originate in the reproductive organs but can also involve the urinary or intestinal tracts if endometriosis implantation has occurred there. The presentation and physical appearance of endometriosis is extremely variable and can be characterized by a chronic intraperitoneal inflammatory process and adhesions. The only definitive diagnostic technique is laparoscopy.To review current literature on the treatment strategies for endometriosis.Review of Pubmed, Cochrane database and Medline for current review articles and studies regarding the current treatment strategies for endometriosis.Initial treatment is surgical or medical. Medical therapy is often used as a first-line therapy and can also be used in conjunction with those patients who undergo surgical therapy for pain. No medical therapy has proven effective for infertility. Medical therapy consists mostly of hormonal suppressive therapy in which the medication causes a downregulation of the hypothalamus-pituitary-ovarian pathway. Non-steroidal anti-inflammatory drugs and oral contraceptives are often used as an initial approach even without a definitive diagnosis. Progestins, such as oral norethindrone and depot medroxyprogesterone, are effective while using them but have a high recurrence rate. The norgestrol intrauterine device is also quite effective at relieving pain associated with endometriosis, especially pain arising during menses as well as from lesions in the rectovaginal tissue. Gonadotropin-releasing hormone agonists induce a pseudomenopausal state and have significant side effects, such as hot flashes and genital atrophy. 'Add-back' therapy with a progestin has been shown to relieve most of these drug related symptoms. Gonadotropin-releasing hormone agonists are also very effective at relieving symptoms of pain during treatment but are also associated with a high recurrence rate. New drug therapies that are under investigation are aromatase inhibitors and immunomodulators. Furthermore, new delivery systems are being investigated that may also improve the patient response.

To compare tubal anastomosis by robotic system compared with outpatient minilaparotomy.In this retrospective case-control study, women were identified by current procedural terminology code for tubal anastomosis. We included all cases of tubal anastomosis for reversal of a prior tubal ligation by either outpatient minilaparotomy or robotic system technique. Cases performed by laparoscopy without aid of the robot were excluded. Comparisons were based on Fisher's exact, chi(2), and Wilcoxon rank sum tests.There were 26 cases of tubal anastomosis performed with the robot and 41 cases performed by outpatient minilaparotomy. The two groups were comparable in age, body mass index, and parity. Anesthesia time for the robotic technique (median with interquartile range) was 283 (267-290) minutes compared with 205 (170-230) minutes with outpatient minilaparotomy (P.001). Surgical times for the robot and minilaparotomy were 229 (205-252) minutes and 181 (154-202) minutes respectively (P=.001). Hospitalization times, pregnancy, and ectopic pregnancy rates were not significantly different. The robotic technique was more costly. The median difference in costs of the procedures was $1,446 (95% confidence interval $1,112-1,812) (P.001). The time to return to work was significantly shorter in the robotic system group by approximately 1 week (P=.013).Robotic surgery for tubal anastomosis was successfully accomplished without conversion to laparotomy. The robotic technique for tubal anastomosis required significantly prolonged surgical and anesthesia times over outpatient minilaparotomy (Por=.001). Costs were higher with the robotic technique. Return to normal activity was shorter with the robotic technique.

Pelvic sonography is performed commonly in patients who have a clinical diagnosis of pelvic inflammatory disease. Although the study may be normal or sometimes nonspecific, there are various findings that are characteristic of this process. Understanding of the sonographic features of pelvic inflammation, salpingitis, pyosalpinx, tubo–ovarian complex and tubo–ovarian abscess will allow the interpreter to make more specific, clinically useful diagnoses. Sonography also can help to distinguish acute from chronic abnormalities in the fallopian tubes. Correlation of sonography with pelvic CT is important, as CT is ordered with increasing frequency in patients who have unexplained lower abdominal pain.

Since being introduced more than 30 years ago, endovaginal ultrasonography (US) and quantitative testing of serum levels of the beta subunit of human chorionic gonadotropin have become the standard means of establishing the presence of normal intrauterine pregnancy (IUP), failed IUP, and ectopic pregnancy. Appropriate use of these powerful tools requires clear, standardized interpretations based on conservative criteria to protect both the pregnancy and the mother. Since diagnoses are assigned earlier and available medical treatments for ectopic pregnancy and failed IUP are expanding, emphasis must carefully shift toward watchful waiting when the mother is clinically stable and a definitive location for the pregnancy cannot be established with US. To this end and to prevent inadvertent harm to early normal pregnancies, the Society of Radiologists in Ultrasound convened a consensus panel of radiologists, obstetricians, and emergency medicine physicians in 2012 with the goal of reviewing current literature and clinical practices and formulating modern criteria and terminology for the various first-trimester outcomes.

We have recently identified a toll-like receptor (TLR4)-interacting SPA4 peptide encoding amino acids: GDFRYSDGTPVNYTNWYRGE, a shorter region of human surfactant protein-A (SP-A). The SPA4 peptide suppressed lipopolysaccharide-induced inflammation (JPET 2011, Innate Immun 2013). In this report, we examined the structure of synthetic SPA4 peptide in solution by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The CD analysis revealed that the SPA4 peptide is composed of ∼35% beta sheet and

Verrucous carcinoma (VC) is an uncommon, low-grade variant of squamous cell carcinoma. Its benign histologic appearance and indolent course may lead to a delayed diagnosis. We report a rare case of an inverted verrucous carcinoma of the buttock presenting as a slow-growing subcutaneous lesion with a draining sinus and no exophytic component, clinically mimicking abscess and gossypiboma, with magnetic resonance imaging (MRI) and pathologic correlation. Biopsy of the lesional base is mandatory for accurate diagnosis. An enhancement pattern with a convoluted undulating appearance in a subcutaneous lesion displayed on MRI should raise a consideration of inverted VC in the differential diagnosis.

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig VH gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.

This study assessed the effects of a commensal, Chaetogaster limnaei limnaei, and a parasitic trematode, Schistosoma mansoni, on infection patterns and life-history responses in the aquatic snail Biomphalaria glabrata. Prevalence of infection was significantly higher in snails that were devoid of C. limnaei limnaei relative to those that were colonized by the commensal, indicating that the oligochaete may protect the host from trematode infection. This finding appeared to be the direct result of the commensal as opposed to indirect stimulation of the immune system, as hemocyte numbers did not differ between C. limnaei limnaei-colonized and noncolonized snails. Snail growth and reproduction were affected by the presence of C. limnaei limnaei and exposure to S. mansoni. Two-way ANOVA revealed a significant effect of both C. limnaei limnaei presence and trematode exposure on B. glabrata growth over the 5-wk study with C. limnaei limnaei-colonized and parasite-infected snails demonstrating the greatest growth. Snails exposed, but uninfected, by S. mansoni demonstrated the lowest growth regardless of commensal colonization. Chaetogaster limnaei limnaei colonization had no effect on egg production, but S. mansoni-infected snails produced significantly more eggs than individuals from other treatment groups. Survival remained over 85% in all treatment groups. The ecological implications of these results are discussed.

GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain. In this structure, the Rabaptin5 domain is a 90-residue-long helix. At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1. In the C-terminal region, it further assembles into a four-helix bundle tetramer. The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys-698 of human Pg was hypothesized to participate in triggering activation in the SK-Pg complex. Here, we report a study of the Lys-698 to Met substitution in the catalytic domain of Pg (microPg) containing the proteolytic activation-resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (K(D)), the recombinant protein shows a nearly 60-fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 A crystal structure of this mutant microPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys-698 of human Pg plays a functional role in the so-called N-terminal insertion activation mechanism by SK.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.

RAG1 and RAG2 catalyze the initial DNA cleavage steps in V(D)J recombination. Fundamental properties of these proteins remain largely unknown. Here, self-association and conformational properties of murine core RAG1 (residues 384-1008) were examined. As determined by multi-angle laser light scattering measurements, the molecular masses of two predominant core RAG1 species corresponded to dimeric and tetrameric states. Similar results were obtained using a RAG1 fragment containing residues 265-1008, indicating that a non-core portion of RAG1 does not alter the oligomerization states observed for the core region. The fraction of core RAG1 in the tetrameric state increased significantly at lower ionic strengths (0.2 versus 0.5 M NaCl), indicating that this oligomeric form may factor into the physiological function of RAG1. In addition, the secondary structural content of core RAG1, obtained by circular dichroism spectroscopy, demonstrated a significant dependence on ionic strength with a 26% increase in alpha-helical content from 0.2 to 1.0 M NaCl. Together, these results indicate that structural and oligomerization properties of core RAG1 are strongly dependent on electrostatic interactions. Furthermore, the secondary structure of core RAG1 changes upon binding to DNA, with larger increases in alpha-helical content upon binding to the recombination signal sequence (RSS) as compared with non-sequence-specific DNA. As shown by electrophoretic mobility shift assays, higher order oligomeric forms of core RAG1 bound to the canonical RSS. Furthermore, core RAG2 (residues 1-387) formed complexes with multimeric RAG1 species bound to a single RSS, providing additional support for the physiological relevance of higher order oligomeric states of RAG1.

Association of RAG2 with the epigenetic marker H3K4me3 through a PHD domain in its noncore region is important for activating the RAG1 component of the RAG recombinase complex for V(D)J recombination. However, immunofluorescence staining of RAG2 expressed in thymocytes suggests RAG2 is localized principally to the nuclear periphery, away from sites of H3K4 modification. To further test for interactions between RAG2 and separate epigenetic markers, we used confocal microscopy to measure full length RAG2 labeled with GFP at its N-terminus (GFP-FL) and expressed in RAG2−/−pro-B cells. Cross-correlation analysis of cells immunostained with antibodies to H3K4me3 and H3K9me3 showed that RAG2 colocalization with H3K4me3 was significantly greater than with H3K9me3. Furthermore, blocking histone demethylation by treating cells with 2,4-pyridinedicarboxylic acid increased colocalization of GFP-FL with H3K4me3, but not its colocalization with H3K9me3. Interestingly, a GFP-labeled T490A mutant of full length RAG2 (GFP-T490A) showed significantly greater colocalization with H3K4me3 than the labeled wild-type RAG2. GFP-labeled RAG2 core alone (GFP-core) exhibited the poorest colocalization with H3K4me3 of the constructs that we measured. Finally, nanoscale resolution using structured illumination microscopy showed that RAG2 was unevenly distributed in the nucleus, containing dispersed RAG2-rich and RAG2-poor regions. Furthermore, RAG2 interactions with H3K4me3 were restricted to interfaces between H3K4me3-enriched puncta and the RAG2. Altogether, these data provide visualization of RAG2 binding to the epigenetic marker H3K4me3, and that Thr490 of RAG2 regulates this interaction.

The in vitro fertilization (IVF) outcomes, including clinical intrauterine gestation rate and live birth rate, between Hispanic and non-Hispanic white women were compared, and there were no differences. Hispanics were more likely to have a diagnosis of tubal factor infertility, whereas non-Hispanic white women were more likely to have endometriosis as their infertility diagnosis.

V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex. Here, we combined limited proteolysis and mass spectrometry methods to identify regions of RAG1 that are sequestered upon association with RAG2. These results show that RAG2 bridges an interdomain boundary in the catalytic region of RAG1. In a second approach, mutation of RAG1 residues within the interdomain boundary were tested for disruption of RAG1:RAG2 complex formation using fluorescence-based pull down assays. The core RAG1 mutants demonstrated varying effects on complex formation with RAG2. Interestingly, two mutants showed opposing results for the ability to interact with core versus full length RAG2, indicating that the non-core region of RAG2 participates in binding to core RAG1. Significantly, all of the RAG1 interdomain mutants demonstrated altered stoichiometries of the RAG complexes, with an increased number of RAG2 per RAG1 subunit compared to the wild type complex. Based on our results, we propose that interaction of RAG2 with RAG1 induces cooperative interactions of multiple binding sites, induced through conformational changes at the RAG1 interdomain boundary, and resulting in formation of the DNA cleavage active site.

V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384-1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topologically independent domains within core RAG1, referred to as the central domain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.

Zinc proteins involved in the control of gene expression include two types which contain zinc binuclear clusters; fungal transcription factors containing DNA binding domains organized around a Zn2Cys6 cluster and the V(D)J recombination-activating proteins, RAG1's, of higher eukaryotes in which the dimerization domains contain a Zn2Cys5His2 cluster. Two solution structures of Zn2Cys6 cluster domains are compared along with the crystal structure of the dimerization domain of RAG1. The dimerization domain of RAG1 contains four zinc ions and is organized around a zinc finger, a zinc RING finger and a zinc binuclear cluster. Protein folds stabilized by multiple zinc sites are discussed.

Thermal data supplied by manufacturers and used as a benchmark in a comparison of the thermal performance of different packages must be treated with caution, as the results are specific to the measurement environment of the supplier. For this reason, calculations of expected component thermal performance based on these data can be erroneous. This paper is an analysis of the discrepancies observed in thermal test results owing to small changes in the exact implementation of the measurements and in the calibration of the device temperature-sensitive parameter (diode Vf). It concludes with recommendations for ensuring more accurate and reproducible thermal resistance measurements.

The constellation of symptoms that has come to be known as multiple chemical sensitivities or idiopathic environmental intolerance is increasingly recognized, although the definition is elusive and its pathogenesis as a distinct entity is unconfirmed. The mechanisms that have been proposed include altered immune function, neurogenic inflammation, neurologic sensitization, and conditioned reflexes and genetic differences in drug metabolism enzymes or factors involved in the generation or response to oxidative stress. To date, no chemically related mechanism has been established.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together from a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

Thermal data for devices provided in manufacturers' data sheets are measured under idealized conditions and are not adequate to predict accurately junction temperature under other conditions. A validated model for the device, which can be employed in a variety of environments, is therefore required. This paper reports on the experimental and simulation work carried out to validate the thermal models for 180 and 224 pin cavity up ceramic pin grid array packages. The thermal test apparatus provides repeatable thermal resistance measurements and the known boundary conditions that are required for ease of simulation.

Outcomes of older adults referred for care management and mental health services through the senior reach gatekeeper model of case finding were examined in this study and compared with the Spokane gatekeeper model. Colorado Senior Reach and the Mid-Kansas Senior Outreach (MKSO) programs are the two Senior Reach Gatekeeper programs modeled after the Spokane program, employing the same community education and gatekeeper model and with mental health treatment for elderly adults in need of support. The three mature programs were compared on seniors served, isolation, and depression ratings. Nontraditional community gatekeepers were trained and referred seniors in need. Findings indicate that individuals served by the two Senior Reach Gatekeeper programs demonstrated significant improvements. Isolation indicators such as social isolation decreased, and depression symptoms and suicide ideation also decreased. These findings for two Senior Reach Gatekeeper programs demonstrate that the gatekeeper approach to training community partners worked in referring at-risk seniors in need, in meeting their needs, and in having a positive impact on their lives.Keywords: nontraditional referrals; isolation; mental health services; depression symptomsOlder adults confront numerous challenges that can seriously affect their lives and their mental health. Issues such as retirement, death of a loved one, changes in health, relocation, and adjusting to other life changes are examples of the conditions and life circumstances that older adults experience (Raschko, 1991). It would seem that older adults would reach out to professionals and seek services, but we know that many do not seek assistance and may be rendered incapable of asking for help (Florio, Jensen, Hendryx, Raschko, & Mathieson, 1998). They may fear the stigma attached to asking for help (Raschko, 1991), or systems may be confusing, lack coordination of services or funding, and be fragmented when the seniors are seeking services (Bartels et al., 2004).In 1978, the Spokane Mental Health, Elder Services Division sought to overcome these barriers by developing a community network of trained persons called "gatekeepers" who could make referrals for older adults they identified as at risk. The gatekeeper model (see Florio et al., 1996, for a complete description) was created to bring at-risk older adults to the attention of professionals who could provide outreach through a care management program.In 2006, the Jefferson Center for Mental Health, the Mental Health Center Serving Boulder and Broomfield Counties, and the Seniors' Resource Center partnered to create a program modeled after the Spokane Gatekeeper program. Previous studies of the gatekeeper model had identified the need for mental health services for these at- risk older adults and the need for more research on this model. The new program incorporated those components and is known as Colorado Senior Reach using the senior reach gatekeeper model.In 2009, the Mental Health Association of South Central Kansas, United Way of the Plains, and several community partners initiated the MKSO project in Wichita, Kansas to replicate the senior reach gatekeeper model. This replication included many of the data elements used in Spokane and Colorado and was modeled closely after the Colorado Senior Reach program elements and fidelity requirements. It also included as a new study element, elder abuse, and prevention strategies as a program area.THE COLORADO SENIOR REACH AND MID- KANSAS SENIOR OUTREACH PROGRAMSFollowing the lead of the Spokane gatekeeper model, the mission of Colorado Senior Reach and Mid-Kansas Senior Outreach (MKSO) programs is to support the well-being and independence of seniors by educating the community on how to identify and refer isolated, at-risk older adults who may benefit from a model of care management, mental health services, and referral assistance.A central aspect of the Colorado Senior Reach program and the MKSO program was to implement the gatekeeper model with fidelity to the Spokane, Washington program that had sought to improve several key outcomes of seniors' lives. …

We have identified a new recurrent reciprocal translocation between chromosome 3 and 12 with breakpoints at bands 3q26 and 12p13, t(3;12)(q26;p13) in the malignant cells from five patients with acute transformation of myelodysplastic syndrome or blast crisis of chronic myelogenous leukemia. t(3;12)(q26;p13) appears as a rare but nonrandom event present in various myeloid leukemia subtypes, which is frequently associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and a very poor prognosis. Here, we report the molecular cytogenetic analysis of the t(3;12). Fluorescence in situ hybridization results indicate that the 3q26 breakpoints are quite heterogeneous and occur 5′ of MDS1, 3′ of EVI1, or between MDS1 and EVI1. Our results are very similar to those observed in other 3q26 rearrangements in which breakpoints were shown to occur over considerable distances 5′ and 3′ of EVI1. Fluorescence in situ hybridization investigations proved that, in three myelodysplastic syndrome cases with t(3;12)(q26;p13), the 12p 13 breakpoint occurred within the TEL gene.

To identify Chlamydia trachomatis antigens associated with tubal factor infertility and acute infection.A C trachomatis proteome array was used to compare antibody profiles among women with tubal factor infertility, normal fertility, and acute C trachomatis infection.Thirteen immunodominant antigens reacted with 50% or more sera from all women (n=73). Six C trachomatis antigens were uniquely recognized in women with tubal factor infertility. Combining fragmentation of the six antigens with serum sample dilution, chlamydial antigens HSP60, CT376, CT557, and CT443 could discriminate between women with tubal factor infertility and women with normal fertility with a sensitivity of 63% (95% confidence interval [CI] 0.41-0.77) and specificity of 100% (95% CI 0.91-1), respectively. These antigens were designated as tubal factor infertility-associated antigens. However, these tubal factor antigens were unable to distinguish tubal factor infertility patients from those with acute infection. A combination of CT875 and CT147 distinguished women with acute infection from all other C trachomatis-exposed women with a detection sensitivity of 63% (95% CI 0.41-0.77) and specificity of 100% (95% CI 0.95-1), respectively. Thus, CT875 and CT147 were designated as acute infection-associated antigens.A sequential screening of antibodies against panels of C trachomatis antigens can be used to identify women with tubal factor infertility and acute C trachomatis infection.II.

OBJECTIVES : This study aimed to examine the risk factors for prevalence and incidence of pelvic organ prolapse (POP) in whites, Hispanics, and blacks. METHODS : This is a secondary analysis of the Women's Health Initiative (WHI) Estrogen plus Progestin Clinical Trial (E + P). Of the original E + P trial population of 16,608, 12,667 women (78.3%; 11,194 whites, 804 blacks, and 669 Hispanics) were included in the final study sample and evaluated during the 5-year period. The outcomes evaluated were any prolapse (WHI prolapse grades 1-3) and WHI prolapse grade 2 or 3. Descriptive analyses, logistic regression, and proportional hazard modeling were performed. RESULTS : Increasing parity correlates with increasing WHI prolapse grades (0-3) in whites and blacks but not Hispanics. The incidence of grade 2 or 3 POP increased by 250% in white women with 1 child (hazard ratio [HR], 2.50; 95% confidence interval [CI], 1.68-3.71) in comparison to nulliparous women and grew with higher parity. For blacks, a weak association between the parity and grade 2 or 3 POP was noted only in women who had 5 or more kids (HR, 10.41; 95% CI, 1.38-78.77). Blacks were less likely (HR, 0.53; 95% CI, 0.40-0.71) to develop grade 2 or 3 POP compared with whites. For grade 2 or 3 POP, age was found to be a risk factor in whites (odds ratio [OR], 1.03; 95% CI, 1.02-1.04) only and body mass index (≥25 kg/m

The DNA binding domain of the GAL4 transcription factor from yeast is located in the N-terminal 60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, Zn2C6, with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 30:11292-11302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UASG, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn2 and Zn1 forms of the N-terminal 149 residues of GAL4, GAL4(149*), for UASG DNA sequences. We show that Cd2- and Zn1GAL4(149*) bind to UASG DNA with 2-fold and 4-8-fold lower affinities than Zn2GAL4(149*), respectively. Thus, the metal species and the number of metal ions bound have measurable effects on the specific DNA binding affinity of GAL4, but these differences are small in comparison to the ratio, > 10(3) under some conditions, that characterizes the specific to nonspecific DNA binding affinities of the N-terminal fragments of GAL4. A shorter N-terminal fragment, GAL4(62*), although it continues to recognize the UASG sequence with a high degree of specificity, binds with 1,000-2,000-fold lower affinity than does Zn2GAL4(149*). Gel retardation titrations of a DNA containing 2 UASG sites with increasing concentrations of GAL4(62*) generate a series of 4 retarded bands in contrast to 2 retarded bands formed when the same DNA is titrated with GAL4(149*). These data suggest that GAL4(62*) binds to the UASG sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UASG DNA cooperatively as a dimer. The approximately 10(3) lower affinity of GAL4(62*) for the UASG DNA can be accounted for by its failure to form dimers in solution. Zn2-, Zn1-, or Cd2GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UASG-containing DNA restriction fragments. Analysis of the data suggests that all 3 proteins cause a 26 degrees angle of bend in the DNA when bound to 1 UASG site and 45 degrees when bound to 2 tandem UASG sites. The same assay shows that GAL4(62*) does not induce significant bending of the UASG DNA sequences. Thus, the additional subdomains found in the larger polypeptide fragment, GAL4(149*), must exert an additional force on the DNA either through direct contacts with the DNA or indirectly through altered protein conformation.

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.

The introduction of American mink (Neovison vison; hereafter mink) into Europe has had severe impacts on many native wildlife species, including the water vole (Arvicola amphibius) in mainland Britain. Although trapping has been widely used to attempt to control mink, managers have little direct evidence of its effect on mink density or distribution, particularly where immigration of mink from nearby areas is inevitable. Such evidence is needed to justify the use of lethal methods in conservation policy. During 2006–2010 we removed mink from the River Monnow Catchment in western Britain, using track-recording rafts to monitor continuously for mink presence, guiding a strategic trapping effort. The area monitored and trapped was increased in stages, from a core sub-catchment with 109 km of water-course in 2006, to a 421-km2 catchment with 203 km of water-course in 2009. In each successive sub-catchment, mink detection and capture rates declined rapidly to near-zero levels after trapping began. Detections and captures showed seasonal peaks in every year corresponding to known dispersal periods, but also declined steadily from year to year, with increasing periods in which we did not detect mink. These results suggested that each sub-catchment was cleared of mink within a few months, with subsequent captures attributable to immigration. On average, we detected each mink 5.1 times before capture (daily probability of detection = 0.059 per mink and raft), and trapped them 3.4 days after deploying traps in response. On average, mink entering the area were likely to have been present for less than 13 days before capture. Water voles had been extinct in the Monnow Catchment since the 1980s. During 2006–2008 (starting 6 months after mink trapping commenced), we released 700 captive-bred water voles into the treatment area to re-establish a wild population. Persistence of this population through the 4 years of the project was considered indicative of effective mink control. This study demonstrates that, even in a mainland context, a systematic trapping strategy can have a substantial impact on the density and distribution of a damaging species, in this case allowing the restoration of a native prey species. © 2013 The Wildlife Society

Background The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction. Results Using a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain (CND; residues 87-217). Conclusions We show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein.

The predictive value of serum beta hCG level for fetal cardiac motion and pregnancy outcome after IVF was evaluated. The serum hCG level 12 days after ET is a useful predictor of subsequent presence of fetal cardiac activity and live birth and may assist clinicians in counseling patients regarding their IVF outcome.

Objective The objective of the study was to assess antibodies against Chlamydia trachomatis heat shock proteins (HSP) in patients with tubal factor infertility (TFI), infertility controls (IFC), and fertile controls (FC). HSPs assist organisms in surviving caustic environments such as heat. Study design Twenty-one TFI, 15 IFC, and 29 FC patients were enrolled after laparoscopic tubal assessment. The titers of antibodies against C trachomatis organisms and 14 chlamydial HSPs were compared among the 3 groups. Results TFI patients developed significantly higher levels of antibodies against C trachomatis and specifically recognizing chlamydial HSP60 and caseinolytic protease (Clp) P, a subunit of the ATP-dependent Clp protease complex involved in the degradation of abnormal proteins. Conclusion In addition to confirming high titers of antibodies against C trachomatis organisms and HSP60 in TFI patients, we identified a novel link of TFI with anti-ClpP antibodies. These findings may provide useful information for developing a noninvasive screening test for TFI and constructing subunit anti- C trachomatis vaccines.