The allohexaploid bread wheat genome consists of three closely related subgenomes (A, B, and D), but a clear understanding of their phylogenetic history has been lacking. We used genome assemblies of bread wheat and five diploid relatives to analyze genome-wide samples of gene trees, as well as to estimate evolutionary relatedness and divergence times. We show that the A and B genomes diverged from a common ancestor ~7 million years ago and that these genomes gave rise to the D genome through homoploid hybrid speciation 1 to 2 million years later. Our findings imply that the present-day bread wheat genome is a product of multiple rounds of hybrid speciation (homoploid and polyploid) and lay the foundation for a new framework for understanding the wheat genome as a multilevel phylogenetic mosaic.

OBJECTIVE

Technologic advances have led to increased clinical use of higher-sized fractions of radiation dose and higher total doses. How these modify the pathways involved in tumor cell death, normal tissue response, and signaling to the immune system has been inadequately explored. Here we ask how radiation dose and fraction size affect antitumor immunity, the suppression thereof, and how this might relate to tumor control.

METHODS

Mice bearing B16-OVA murine melanoma were treated with up to 15 Gy radiation given in various-size fractions, and tumor growth followed. The tumor-specific immune response in the spleen was assessed by interferon-γ enzyme-linked immunospot (ELISPOT) assay with ovalbumin (OVA) as the surrogate tumor antigen and the contribution of regulatory T cells (Tregs) determined by the proportion of CD4(+)CD25(hi)Foxp3(+) T cells.

RESULTS

After single doses, tumor control increased with the size of radiation dose, as did the number of tumor-reactive T cells. This was offset at the highest dose by an increase in Treg representation. Fractionated treatment with medium-size radiation doses of 7.5 Gy/fraction gave the best tumor control and tumor immunity while maintaining low Treg numbers.

CONCLUSIONS

Radiation can be an immune adjuvant, but the response varies with the size of dose per fraction. The ultimate challenge is to optimally integrate cancer immunotherapy into radiation therapy.

Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.

The discovery of audiovisual mirror neurons in monkeys gave rise to the hypothesis that premotor areas are inherently involved not only when observing actions but also when listening to action-related sound. However, the whole-brain functional formation underlying such "action-listening" is not fully understood. In addition, previous studies in humans have focused mostly on relatively simple and overexperienced everyday actions, such as hand clapping or door knocking. Here we used functional magnetic resonance imaging to ask whether the human action-recognition system responds to sounds found in a more complex sequence of newly acquired actions. To address this, we chose a piece of music as a model set of acoustically presentable actions and trained non-musicians to play it by ear. We then monitored brain activity in subjects while they listened to the newly acquired piece. Although subjects listened to the music without performing any movements, activation was found bilaterally in the frontoparietal motor-related network (including Broca's area, the premotor region, the intraparietal sulcus, and the inferior parietal region), consistent with neural circuits that have been associated with action observations, and may constitute the human mirror neuron system. Presentation of the practiced notes in a different order activated the network to a much lesser degree, whereas listening to an equally familiar but motorically unknown music did not activate this network. These findings support the hypothesis of a "hearing-doing" system that is highly dependent on the individual's motor repertoire, gets established rapidly, and consists of Broca's area as its hub.

BACKGROUND

The dual challenges to tuberculosis (TB) control of HIV infection and multidrug resistance are particularly pressing in South Africa. Conventional methods for detecting Mycobacterium tuberculosis drug resistance take weeks to months to produce results. Rapid molecular testing for drug resistance is available but has not been implemented in high-TB-burden settings.

OBJECTIVE

To assess the performance and feasibility of implementation of a commercially available molecular line-probe assay for rapid detection of rifampicin and isoniazid resistance.

METHODS

We performed the assay directly on 536 consecutive smear-positive sputum specimens from patients at increased risk of multidrug-resistant (MDR) TB in a busy routine diagnostic laboratory in Cape Town, South Africa. Results were compared with conventional liquid culture and drug susceptibility testing on solid medium.

RESULTS

Overall, 97% of smear-positive specimens gave interpretable results within 1-2 days using the molecular assay. Sensitivity, specificity, and positive and negative predictive values were 98.9, 99.4, 97.9, and 99.7%, respectively, for detection of rifampicin resistance; 94.2, 99.7, 99.1, and 97.9%, respectively, for detection of isoniazid resistance; and 98.8, 100, 100, and 99.7%, respectively, for detection of multidrug resistance compared with conventional results. The assay also performed well on specimens that were contaminated on conventional culture and on smear-negative, culture-positive specimens.

CONCLUSIONS

This molecular assay is a highly accurate screening tool for MDR TB, which achieves a substantial reduction in diagnostic delay. With overall performance characteristics that are superior to conventional culture and drug susceptibility testing and the possibility for high throughput with substantial cost savings, molecular testing has the potential to revolutionize MDR TB diagnosis.

BACKGROUND

Whether bone markers have prognostic value in patients with bone metastases is unknown. We investigated this question in patients with bone metastases secondary to prostate cancer and to non-small-cell lung cancer (NSCLC) and other solid tumors assigned to the placebo arms of two phase III trials of zoledronic acid.

METHODS

Levels of the urinary bone resorption marker N-telopeptide and the serum bone formation marker bone-specific alkaline phosphatase were assessed every 3 months for patients with prostate cancer (n = 203) or NSCLC or other solid tumors (n = 238) and were categorized as low or high. Patients were monitored for skeletal-related events, bone disease progression, and death. The relative risks (RRs) and 95% confidence intervals (CIs) for these outcomes were estimated for patients with high versus low levels of each marker using intensity-based multiple event and Cox regression models. All statistical tests were two-sided.

RESULTS

In each disease group and overall, high levels of each marker at the beginning of the study were statistically significantly associated with an increased risk of negative outcomes. Use of recent marker assessments as time-dependent covariates gave even greater prognostic significance. High N-telopeptide levels were a stronger prognostic indicator of negative outcomes than bone-specific alkaline phosphatase levels. In recent assessments, patients with high N-telopeptide levels had an increased relative risk of skeletal-related events (prostate cancer, RR = 3.25, 95% CI = 2.26 to 4.68, P<.001; NSCLC and other solid tumors, RR = 1.79, 95% CI = 1.15 to 2.79, P = .010), disease progression (prostate cancer, RR = 2.02, 95% CI = 1.48 to 2.74, P<.001; NSCLC and other solid tumors, RR = 1.91, 95% CI = 1.16 to 3.15, P = .011), and death (prostate cancer, RR = 4.59, 95% CI = 2.82 to 7.46, P<.001; NSCLC and other solid tumors, RR = 2.67, 95% CI = 1.85 to 3.85, P<.001) compared with patients with low N-telopeptide levels.

CONCLUSIONS

Baseline and recent bone marker levels were predictive of negative clinical outcomes in patients with bone metastases secondary to prostate cancer and to NSCLC and other solid tumors. N-telopeptide levels were more consistent prognostic indicators than bone-specific alkaline phosphatase for all tumor types, reflecting the key role of osteolysis in the development of skeletal complications.

The first leukocytes that arise in the development of vertebrate embryos are the primitive macrophages, which differentiate in the yolk sac and then quickly invade embryonic tissues. These macrophages have been considered to constitute a separate lineage, giving rise to no other cell type. Using an in vivo photoactivatable cell tracer in the transparent zebrafish (Danio rerio) embryo, we demonstrated that this lineage also gave rise to an equal or higher number of neutrophilic granulocytes. We were surprised to find that the differentiation of these primitive neutrophils occurs only after primitive myeloid progenitors have dispersed in the tissues. By 2 days after fertilization, these neutrophils have become the major leukocyte type found wandering in the epidermis and mesenchyme. Like the primitive macrophages, all primitive and larval neutrophils express PU.1 and L-plastin and they are highly attracted to local infections, yet only a small fraction of them phagocytose microbes, and to a much lesser extent per cell than the macrophages. They are also attracted to variously stressed or malformed tissues, suggesting a wider role than antimicrobial defense.

The organization of collagen fibrils in the human cornea and sclera was studied by scanning electron microscopy, after digestion of cellular elements by sodium hydroxide, and by conventional transmission electron microscopy. The collagen fibrils in the cornea had a uniform diameter of about 25 nm. In Bowman's layer, individual collagen fibrils were interwoven densely to form a felt-like sheet. In the stroma, most of the collagen fibrils ran abreast in lamellae, with varying widths and thickness. These lamellae were arranged basically parallel to the corneal surface but often communicated with those of adjacent layers by interchanging their fibrils. In the innermost stromal region adjacent to Descemet's membrane, collagen fibrils were oriented in various directions and interlaced, forming loose fibrillar networks. The sclera, however, was composed of collagen fibrils with various diameters ranging from 25-230 nm. Although these collagen fibrils formed bundles, they were not parallel but were entangled in individual bundles. These collagen bundles varied in width and thickness, often gave off branches, and intertwined with each other.

The rat basophilic leukemia (RBL) cell lines were cloned and the various sublines compared for their chromosome number, IgE-mediated histamine release and for IgE surface receptors. It was found that cell lines started from tumors at different times vary in both their chromosome number and their ability to release histamine by an IgE-mediated reaction. RBL-I and III have approximately 44 chromosomes and did not respond to an IgE-mediated reaction. RBL-II and RBL-IV have 68-73 chromosomes and showed moderate levels of histamine release (percent release mean = 5 +/- 2 and 10 +/- 4, respectively). The cloning of the RBL-IV line resulted in some sublines which were excellent histamine releasers (range 39-100%) and some which were relatively refractory (less than 10%) to IgE-mediated histamine release. These clones did not differ significantly in chromosome number. Recloning the releasing lines gave rise to poor releasers, whereas the recloning of poor releasers did not produce good releasers indicating that the mutational drift in culture is toward loss of histamine-releasing capacity. The number of IgE receptors and the rate of IgE association and dissociation were similar for the different cell lines. The study failed to disclose significant molecular weight differences in the IgE receptor from the various clones and sublines indicating that the failure to release probably does not reside in the receptor. The various cloned sublines are phenotypically stable, and the isolation of excellent histamine-releasing sublines are useful for studies of the complex phenomenon of the histamine release.

BACKGROUND

Prostate specific membrane antigen (PSM) is a membrane-bound antigen that is highly specific for benign and malignant prostate epithelial cells. Its expression in high grade prostatic intraepithelial neoplasia (PIN) has not been compared with that in prostate carcinoma.

METHODS

The authors performed an immunohistochemical study of representative sections from 184 radical prostatectomies from previously untreated patients with pathologic stage T2N0M0 adenocarcinoma treated at the Mayo Clinic between 1987 and 1991. Affinity-purified monoclonal antibody 7E11-5.3 directed against PSM was employed at a concentration of 20 microg/mL overnight. For comparison, serial sections in each case were stained with prostate specific antigen (PSA). Staining for all antibodies was performed using the streptavidin-biotin method. For each case, the percentage of immunoreactive cells in benign epithelium, PIN, and adenocarcinoma was estimated in increments of 10%. Cox proportional hazards models were used to identify the risk of carcinoma recurrence according to the number of immunoreactive PIN or cancer cells for PSM and PSA; the date of radical prostatectomy was used as the starting time, and serum PSA (biochemical) failure or clinical failure was the event. PSA biochemical failure was defined as serum PSA>> 0.2 ng/mL at least 30 days after surgery.

RESULTS

Intense cytoplasmic immunoreactivity for PSM was observed in the benign and neoplastic epithelial cells in all cases (100% of cases staining). The number of cells staining was lower in benign epithelium and PIN than in adenocarcinoma (69.5+/-17.3% [range, 20-90%] vs. 77.9+/-13.2% [range, 30-100%] vs. 80.2+/-13.7% [range, 30-100%], respectively). With rare exceptions, basal cells were negative, and there was no immunoreactivity of the prostate stroma, urothelium, or vasculature. Adenocarcinoma gave the most intense and extensive staining, and the highest grades of adenocarcinoma (Gleason primary patterns 4 and 5) showed staining in virtually every cell; there was greater heterogeneity of staining in lower grades of adenocarcinoma. By contrast, PSA immunoreactivity was more intense and extensive in benign epithelium than in PIN and adenocarcinoma. The number of immunoreactive PIN or cancer cells for PSM and PSA was not predictive of PSA biochemical or clinical failure as defined in this study.

CONCLUSIONS

PSM was expressed in all cases of prostate adenocarcinoma, with the greatest extent and intensity observed in the highest grades. The expression increased incrementally from benign epithelium to high grade PIN or adenocarcinoma. Conversely, PSA showed the greatest staining in benign epithelium, with decreased expression incrementally from benign epithelium to high grade PIN or adenocarcinoma. Expression of PSM is clinically useful for the identification of prostate epithelium, particularly PIN or adenocarcinoma, and its expression is regulated independent of PSA. The number of PSM immunoreactive cells was not predictive of recurrence, most likely because of the presence of abundant immunoreactivity in most cases, or because of differential expression in primary and metastatic disease.

OBJECTIVE

To assess the clinical effectiveness and cost-effectiveness of buprenorphine maintenance therapy (BMT) and methadone maintenance therapy (MMT) for the management of opioid-dependent individuals.

METHODS

Major electronic databases were searched from inception to August 2005. Industry submissions to the National Institute for Health and Clinical Excellence were accessed.

METHODS

The assessment of clinical effectiveness was based on a review of existing reviews plus an updated search for randomised controlled trials (RCTs). A decision tree with Monte Carlo simulation model was developed to assess the cost-effectiveness of BMT and MMT. Retention in treatment and opiate abuse parameters were sourced from the meta-analysis of RCTs directly comparing flexible MMT with flexible dose BMT. Utilities were derived from a panel representing a societal perspective.

RESULTS

Most of the included systematic reviews and RCTs were of moderate to good quality, and focused on short-term (up to 1-year follow-up) outcomes of retention in treatment and the level of opiate use (self-report or urinalysis). Most studies employed a trial design that compared a fixed-dose strategy (i.e. all individuals received a standard dose) of MMT or BMT and were conducted in predominantly young men who fulfilled criteria as opiate-dependent or heroin-dependent users, without significant co-morbidities. RCT meta-analyses have shown that a fixed dose of MMT or BMT has superior levels of retention in treatment and opiate use than placebo or no treatment, with higher fixed doses being more effective than lower fixed doses. There was evidence, primarily from non-randomised observational studies, that fixed-dose MMT reduces mortality, HIV risk behaviour and levels of crime compared with no therapy and one small RCT has shown the level of mortality with fixed-dose BMT to be significantly less than with placebo. Flexible dosing (i.e. individualised doses) of MMT and BMT is more reflective of real-world practice. Retention in treatment was superior for flexible MMT than flexible BMT dosing but there was no significant difference in opiate use. Indirect comparison of data from population cross-sectional studies suggests that mortality with BMT may be lower than that with MMT. A pooled RCT analysis showed no significant difference in serious adverse events with MMT compared with BMT. Although treatment modifier evidence was limited, adjunct psychosocial and contingency interventions (e.g. financial incentives for opiate-free urine samples) appeared to enhance the effects of both MMT and BMT. Also, MMT and BMT appear to be similarly effective whether delivered in a primary care or outpatient clinic setting. Although most of the included economic evaluations were considered to be of high quality, none used all of the appropriate parameters, effectiveness data, perspective and comparators required to make their results generalisable to the NHS context. One company (Schering-Plough) submitted cost-effectiveness evidence based on an economic model that had a 1-year time horizon and sourced data from a single RCT of flexible-dose MMT compared with flexible-dose BMT and utility values obtained from the literature; the results showed that for MMT vs no drug therapy, the incremental cost-effectiveness ratio (ICER) was pound 12,584/quality-adjusted life-year (QALY), for BMT versus no drug therapy, the ICER was pound 30,048/QALY and in a direct comparison, MMT was found to be slightly more effective and less costly than BMT. The assessment group model found for MMT versus no drug therapy that the ICER was pound 13,697/QALY, for BMT versus no drug therapy that the ICER was pound 26,429/QALY and, as with the industry model, in direct comparison, MMT was slightly more effective and less costly than BMT. When considering social costs, both MMT and BMT gave more health gain and were less costly than no drug treatment. These findings were robust to deterministic and probabilistic sensitivity analyses.

CONCLUSIONS

Both flexible-dose MMT and BMT are more clinically effective and more cost-effective than no drug therapy in dependent opiate users. In direct comparison, a flexible dosing strategy with MMT was found be somewhat more effective in maintaining individuals in treatment than flexible-dose BMT and therefore associated with a slightly higher health gain and lower costs. However, this needs to be balanced by the more recent experience of clinicians in the use of buprenorphine, the possible risk of higher mortality of MMT and individual opiate-dependent users' preferences. Future research should be directed towards the safety and effectiveness of MMT and BMT; potential safety concerns regarding methadone and buprenorphine, specifically mortality and key drug interactions; efficacy of substitution medications (in particular patient subgroups, such as within the criminal justice system, or within young people); and uncertainties in cost-effectiveness identified by current economic models.

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.

The destiny of the mitotically active cells of the subventricular zone (SVZ) in adult rodents is to migrate to the olfactory bulb, where they contribute to the replacement of granular and periglomerular neurons. However, these adult neural progenitors also can be mobilized in periventricular white matter and triggered to differentiate into astrocytes and oligodendrocytes in response to lysolecithin-induced demyelination. To mimic the environmental conditions of multiple sclerosis, we assessed the proliferation, migration, and differentiation potential of adult SVZ progenitor cells in response to experimental autoimmune encephalomyelitis (EAE) in mice. Inflammation and demyelination were observed in all mouse brains after EAE induction. EAE induced cell proliferation throughout the brain and especially within the lesions. Proliferating cells were neural progenitors, astrocytes, and oligodendrocyte precursors. EAE enhanced the migration of SVZ-derived neural progenitors to the olfactory bulb and triggered their mobilization in the periventricular white matter. The mobilized cells gave rise to neurons, astrocytes, and oligodendrocytes in the olfactory bulb but essentially to astrocytes and oligodendrocytes in the lesioned white matter. Our data indicate that the adult mouse SVZ is a source of newly generated oligodendrocytes and thus may contribute, along with oligodendrocyte precursors, to the replacement of oligodendrocytes in inflammatory demyelinating diseases of the central nervous system such as multiple sclerosis.

BACKGROUND

Almost two decades since the initiation of the Safe motherhood Initiative, Maternal Mortality is still soaring high in most developing countries. In 2000 WHO estimated a life time risk of a maternal death of 1 in 16 in Sub- Saharan Africa while it was only 1 in 2800 in developed countries. This huge discrepancy in the rate of maternal deaths is due to differences in access and use of maternal health care services. It is known that having a skilled attendant at every delivery can lead to marked reductions in maternal mortality. For this reason, the proportion of births attended by skilled health personnel is one of the indicators used to monitor progress towards the achievement of the MDG-5 of improving maternal health.

METHODS

Cross sectional study which employed quantitative research methods.

RESULTS

We interviewed 974 women who gave birth within one year prior to the survey. Although almost all (99.8%) attended ANC at least once during their last pregnancy, only 46.7% reported to deliver in a health facility and only 44.5% were assisted during delivery by a skilled attendant. Distance to the health facility (OR = 4.09 (2.72-6.16)), discussion with the male partner on place of delivery (OR = 2.37(1.75-3.22)), advise to deliver in a health facility during ANC (OR = 1.43 (1.25-2.63)) and knowledge of pregnancy risk factors (OR 2.95 (1.65-5.25)) showed significant association with use of skilled care at delivery even after controlling for confounding factors.

CONCLUSIONS

Use of skilled care during delivery in this district is below the target set by ICPD + of attaining 80% of deliveries attended by skilled personnel by 2005. We recommend the following in order to increase the pace towards achieving the MDG targets: to improve coverage of health facilities, raising awareness for both men and women on danger signs during pregnancy/delivery and strengthening counseling on facility delivery and individual birth preparedness.

OBJECTIVE

To prospectively evaluate the elastographic appearance of thyroid gland tumors and explore the potential sensitivity and specificity of ultrasonographic (US) elastography for differentiating benign and malignant tumors, with histopathologic analysis as the reference standard.

METHODS

The study was institutional review board approved, and each patient gave written informed consent. Fifty-two thyroid gland lesions (22 malignant, 30 benign) in 31 consecutive patients (six men, 25 women; mean age, 49.7 years +/- 14.7 [standard deviation]) were examined with real-time elastography in the elasticity imaging mode implemented on a clinical US scanner modified for research. In addition, the radiofrequency echo data stored during US were exported from the scanner and used for off-line strain image reconstruction. All elastograms were evaluated for the lesion visibility, relative brightness, and margin regularity and definition by using a four-point scale. In addition, normal thyroid gland tissue and thyroid gland tumor strains were measured on off-line processed elastograms, and the thyroid gland-to-tumor strain ratio (ie, strain index) was calculated. The potential of elastographic criteria for the diagnosis of thyroid gland cancer was evaluated with univariate analysis and multivariate logistic regression. Qualitative variables were compared by using the chi2 test, and quantitative variables were compared by using the Mann-Whitney U test. P < .05 was considered to indicate significance.

RESULTS

A strain index value greater than 4 on off-line processed elastograms was the strongest independent predictor of thyroid gland malignancy (P < .001); this criterion had 96% specificity and 82% sensitivity. Two other elastographic criteria, which were evaluated on real-time elastograms--a margin regularity score higher than 3 (88% specificity, 36% sensitivity) and a tumor area ratio higher than 1 (92% specificity, 46% sensitivity)--also were associated with malignancy (P < .05). However, the usefulness of these criteria was not considered to be high because of their low sensitivity.

CONCLUSIONS

Elastography is a promising imaging technique that can assist in the differential diagnosis of thyroid cancer.

Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the 'genomic context' combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK-HerA superfamily ATPases.

Ten monoclonal antibodies developed against Plasmodium falciparum sporozoites at four institutions were evaluated for use in an enzyme-linked immunosorbent assay (ELISA). Four of the antibodies were eliminated because of their low sensitivity or requirement for high concentrations of capture antibody, while an additional four were rejected because they exhibited cross-reactivity with P. berghei sporozoites. Of the two remaining monoclonal antibodies, that designated 2A10 had the highest sensitivity, a requirement for lower concentrations of capture antibody, and had been tested successfully against sporozoites from a wider range of geographical areas than the others. Use of this monoclonal antibody in a standardized ELISA method gave a test ten times more sensitive than previously reported for P. falciparum sporozoites and its detection limit was less than 100 sporozoites per mosquito.

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A(2A) receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66-88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse alpha(2A)-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.

1. The magnetic field generated by a brief current in a 9 cm diameter flat circular coil varies rapidly with time and when applied over the human scalp it is capable of exciting motor structures subserving the small hand muscles. With a peak magnetic field at the centre of the coil of 0.9-1.6 Tesla, single scalp stimuli produced twitches in the right abductor digiti minimi (a.d.m.), first dorsal interosseous (f.d.i.) and adductor pollicis muscles. Compound muscle action potentials (c.m.a.p.s) and single motor units from these muscles were recorded. 2. The twitch force and corresponding c.m.a.p. were greatly enhanced by voluntary background contraction of the muscle and depended on the direction and intensity of the magnetic field. Response amplitude was maximal with the stimulating coil centred near the vertex but precise coil position was not critical. 3. When the orthodromic volley set up in the peripheral nerve by magnetic stimulation of the brain collided with a maximal antidromic volley set up by stimulation at the wrist, remaining electromyographic (e.m.g.) activity gave evidence of multiple firing of some spinal motoneurones, provided that the muscle exerted a slight voluntary background contraction and the stimulus intensity was above threshold for relaxed muscle. 4. When the muscle changed from total relaxation to a slightly contracted state the onset latency of the c.m.a.p. was shortened by about 3 ms without further change when the background contraction increased. In slightly contracted muscle, c.m.a.p. onset latency was little affected over a wide range of stimulus intensities and was unaffected by the position of the stimulating coil within an area of 6 X 6 cm over the vertex. 5. Single motor units, recorded with needle electrodes in the a.d.m. or f.d.i. muscle, could be caused to discharge at a constant latency by threshold brain stimuli. Different single motor units had latencies varying from 22.4 to 32.1 ms. The same motor unit was activated from different coil positions up to 7 cm apart. These motor units had the lowest thresholds for voluntary activation. Stronger stimuli caused the same motor unit to discharge 1.5 ms earlier. 6. The threshold for excitation of pathways to the a.d.m. muscle was reduced by voluntary contraction of the contralateral a.d.m. and by contraction of the ipsilateral f.d.i. muscles. Using these procedures, the c.m.a.p. of totally relaxed a.d.m. muscle showed a more than 2-fold amplitude increase, a shortening of onset latency to a similar degree as with contraction of the muscle itself, and single motor units in the a.d.m. muscle discharged 1.5 ms earlier.(ABSTRACT TRUNCATED AT 400 WORDS)

In cardiac muscle, release of activator calcium from the sarcoplasmic reticulum occurs by calcium- induced calcium release through ryanodine receptors (RyRs), which are clustered in a dense, regular, two-dimensional lattice array at the diad junction. We simulated numerically the stochastic dynamics of RyRs and L-type sarcolemmal calcium channels interacting via calcium nano-domains in the junctional cleft. Four putative RyR gating schemes based on single-channel measurements in lipid bilayers all failed to give stable excitation-contraction coupling, due either to insufficiently strong inactivation to terminate locally regenerative calcium-induced calcium release or insufficient cooperativity to discriminate against RyR activation by background calcium. If the ryanodine receptor was represented, instead, by a phenomenological four-state gating scheme, with channel opening resulting from simultaneous binding of two Ca2+ ions, and either calcium-dependent or activation-linked inactivation, the simulations gave a good semiquantitative accounting for the macroscopic features of excitation-contraction coupling. It was possible to restore stability to a model based on a bilayer-derived gating scheme, by introducing allosteric interactions between nearest-neighbor RyRs so as to stabilize the inactivated state and produce cooperativity among calcium binding sites on different RyRs. Such allosteric coupling between RyRs may be a function of the foot process and lattice array, explaining their conservation during evolution.

The authors gave the CES-D, a self-report depression symptom scale, to 515 people drawn from a longitudinal community survey. The subjects were also interviewed using the Schedule for Affective Disorders and Schizophrenia (SADS). From the information collected on the SADS, the subjects were given diagnoses based on Research Diagnostic Criteria. The results indicate a modest relationship between self-reported symptoms of depression and the diagnosis of a major or minor depression. However, the groups defined as "cases" by such reports also include many people with other diagnoses or with no diagnoses at all. Thus, symptom scales are useful for the screening of depressed persons in research studies but are only rough indicators of clinical depression in the community.

One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase. Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control. The major limitation of this system is the efficiency with which DNA is introduced into the cell. Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80%. To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids. When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells. However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines. Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.