The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant Staphylococcus aureus (MRSA) strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 microg of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 microg of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was>> or =8 mg of vancomycin per liter and>> or =8 microg teicoplanin per ml or>> or =12 microg of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-)glycopeptide-intermediate S. aureus compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (n = 48) and MRSA (n = 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.

OBJECTIVE

To determine whether hydrogen 1 magnetic resonance (MR) spectroscopic imaging can be used to predict aggressiveness of prostate cancer.

METHODS

All patients gave informed consent according to an institutionally approved research protocol. A total of 123 patients (median age, 58 years; age range, 40-74 years) who underwent endorectal MR imaging and MR spectroscopic imaging between January 2000 and December 2002 were included. MR imaging and spectroscopy were performed by using combined pelvic phased-array and endorectal probe. Water and lipids were suppressed, and phase-encoded data were acquired with 6.2-mm resolution. Voxels in the peripheral zone were considered suspicious for cancer if (Cho + Cr)/Cit was at least two standard deviations above the normal level, where Cho represents choline-containing compounds, Cr represents creatine and phosphocreatine, and Cit represents citrate. Correlation between metabolite ratio and four Gleason score groups identified at step-section pathologic evaluation (3 + 3, 3 + 4, 4 + 3, and>> or =4 + 4) was assessed with generalized estimating equations.

RESULTS

Data from 94 patients were included. Pathologic evaluation was used to identify 239 lesions. Overall sensitivity of MR spectroscopic imaging was 56% for tumor detection, increasing from 44% in lesions with Gleason score of 3 + 3 to 89% in lesions with Gleason score greater than or equal to 4 + 4. There was a trend toward increasing (Cho + Cr)/Cit with increasing Gleason score in lesions identified correctly with MR spectroscopic imaging. Tumor volume assessed with MR spectroscopic imaging increased with increasing Gleason score.

CONCLUSIONS

MR spectroscopic imaging measurement of prostate tumor (Cho + Cr)/Cit and tumor volume correlate with pathologic Gleason score. There is overlap between MR spectroscopic imaging parameters at various Gleason score levels, which may reflect methodologic and physiologic variations. MR spectroscopic imaging has potential in noninvasive assessment of prostate cancer aggressiveness.

To investigate the role of different cysteine residues in bovine rhodopsin, a series of mutants were prepared in which the cysteine residues were systematically replaced by serines. The mutant genes were expressed in monkey kidney cells (COS-1) and the mutant opsins were evaluated for their levels of expression, glycosylation patterns, and ability to form the chromophore characteristic of rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane-embedded cysteines (Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutions of Cys-185 gave rise to a wild-type phenotype. In contrast, substitution of the three intradiscal cysteines (Cys-110, Cys-185, and Cys-187) or single substitution of Cys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal. Thus, of the 10 cysteines in bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essential for the correct tertiary structure of the protein.

A simple disk diffusion test was constructed for detection of IMP-1-type metallo-beta-lactamase-producing gram-negative bacteria. Two Kirby-Bauer disks containing ceftazidime (CAZ) and a filter disk containing a metallo-beta-lactamase inhibitor were used in this test. Several IMP-1 inhibitors such as thiol compounds including 2-mercaptopropionic acid, heavy metal salts, and EDTA were evaluated for this test. Two CAZ disks were placed on a Mueller-Hinton agar plate on which a bacterial suspension was spread according to the method recommended by the National Committee for Clinical Laboratory Standards. The distance between the disks was kept to about 4 to 5 cm, and a filter disk containing a metallo-beta-lactamase inhibitor was placed near one of the CAZ disks within a center-to-center distance of 1.0 to 2.5 cm. For IMP-1-producing strains, the growth-inhibitory zone between the two disks expanded, while no evident change in the shape of the growth-inhibitory zone was observed for CAZ-resistant strains producing serine beta-lactamases such as AmpC or SHV-12. As a result, 2 to 3 microliter of undiluted 2-mercaptopropionic acid or mercaptoacetic acid able to block IMP-1 activity gave the most reproducible and clearest results, and CAZ-resistant strains producing AmpC or extended-spectrum beta-lactamases were distinguishable from IMP-1 producers by this test. A similar observation was made with IMP-1-producing clinical isolates such as Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter spp., and Alcaligenes xylosoxidans. The specificity and sensitivity of this test were comparable to those of PCR analysis using bla(IMP)-specific primers. Therefore, this convenient test would be valuable for daily use in clinical laboratories.

A 10 microm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca(2+) concentration ([Ca(2+)](i)) and changes in [Ca(2+)](i) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin alpha-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau~1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca(2+)](i). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca(2+)](i). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mM Mg(2+) from 20 to 37 degrees C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37 degrees C were used to estimate absolute levels of rod [Ca(2+)](i). All three dyes gave similar values for [Ca(2+)](i) in wild-type rods of 250 +/- 20 nM in darkness and 23 +/- 2 nM after exposure to saturating light. There was no significant difference in dark [Ca(2+)](i) between wild-type and Tralpha-/- animals.

BACKGROUND

Type 2 diabetes mellitus is an increasingly serious health problem among African American women. Consumption of sugar-sweetened drinks was associated with an increased risk of diabetes in 2 studies but not in a third; however, to our knowledge, no data are available on African Americans regarding this issue. Our objective was to examine the association between consumption of sugar-sweetened beverages, weight gain, and incidence of type 2 diabetes mellitus in African American women.

METHODS

A prospective follow-up study of 59,000 African American women has been in progress since 1995. Participants reported on food and beverage consumption in 1995 and 2001. Biennial follow-up questionnaires ascertained new diagnoses of type 2 diabetes. The present analyses included 43,960 women who gave complete dietary and weight information and were free from diabetes at baseline. We identified 2713 incident cases of type 2 diabetes mellitus during 338,884 person-years of follow-up. The main outcome measure was the incidence of type 2 diabetes mellitus.

RESULTS

The incidence of type 2 diabetes mellitus was higher with higher intake of both sugar-sweetened soft drinks and fruit drinks. After adjustment for confounding variables including other dietary factors, the incidence rate ratio for 2 or more soft drinks per day was 1.24 (95% confidence interval, 1.06-1.45). For fruit drinks, the comparable incidence rate ratio was 1.31 (95% confidence interval, 1.13-1.52). The association of diabetes with soft drink consumption was almost entirely mediated by body mass index, whereas the association with fruit drink consumption was independent of body mass index.

CONCLUSIONS

Regular consumption of sugar-sweetened soft drinks and fruit drinks is associated with an increased risk of type 2 diabetes mellitus in African American women. While there has been increasing public awareness of the adverse health effects of soft drinks, little attention has been given to fruit drinks, which are often marketed as a healthier alternative to soft drinks.

Alpha/beta interferons (IFN-alpha/beta) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-alpha/beta responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV(KUN)), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-beta promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNV(KUN) genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-alpha/beta in infected human A549 cells than that detected following wild-type WNV(KUN) infection. Consequently, replication of the WNV(KUN)NS2A/A30P mutant virus in these cells known to be high producers of IFN-alpha/beta was abortive. In contrast, both the mutant and the wild-type WNV(KUN) produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-alpha/beta production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-alpha/betagamma receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV(KUN) and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.

Late-onset Alzheimer's disease (LOAD) is the most common form of dementia in the elderly. The National Institute of Aging-Late Onset Alzheimer's Disease Family Study and the National Cell Repository for Alzheimer's Disease conducted a joint genome-wide association study (GWAS) of multiplex LOAD families (3,839 affected and unaffected individuals from 992 families plus additional unrelated neurologically evaluated normal subjects) using the 610 IlluminaQuad panel. This cohort represents the largest family-based GWAS of LOAD to date, with analyses limited here to the European-American subjects. SNPs near APOE gave highly significant results (e.g., rs2075650, p = 3.2×10(-81)), but no other genome-wide significant evidence for association was obtained in the full sample. Analyses that stratified on APOE genotypes identified SNPs on chromosome 10p14 in CUGBP2 with genome-wide significant evidence for association within APOE ε4 homozygotes (e.g., rs201119, p = 1.5×10(-8)). Association in this gene was replicated in an independent sample consisting of three cohorts. There was evidence of association for recently-reported LOAD risk loci, including BIN1 (rs7561528, p = 0.009 with, and p = 0.03 without, APOE adjustment) and CLU (rs11136000, p = 0.023 with, and p = 0.008 without, APOE adjustment), with weaker support for CR1. However, our results provide strong evidence that association with PICALM (rs3851179, p = 0.69 with, and p = 0.039 without, APOE adjustment) and EXOC3L2 is affected by correlation with APOE, and thus may represent spurious association. Our results indicate that genetic structure coupled with ascertainment bias resulting from the strong APOE association affect genome-wide results and interpretation of some recently reported associations. We show that a locus such as APOE, with large effects and strong association with disease, can lead to samples that require appropriate adjustment for this locus to avoid both false positive and false negative evidence of association. We suggest that similar adjustments may also be needed for many other large multi-site studies.

A new method is proposed to subtract the count of scattered photons from that acquired with a photopeak window at each pixel in each planar image of single-photon emission computed tomography (SPECT). The subtraction is carried out using two sets of data: one set is acquired with a main window centered at photopeak energy and the other is acquired with two subwindows on both sides of the main window. The scattered photons included in the main window are estimated from the counts acquired with the subwindows and then they are subtracted from the count acquired with the main windows. Since the subtraction is performed at each pixel in each planar image, the proposed method has the potential to be more precise than conventional methods. For three different activity distributions in cylinder phantoms, simulation tests gave good agreement between the activity distributions reconstructed from unscattered photons and those from the corrected data.

A popular method for investigating whether stimulus information is present in fMRI response patterns is to attempt to "decode" the stimuli from the response patterns with a multivariate classifier. The sensitivity for detecting the information depends on the particular classifier used. However, little is known about the relative performance of different classifiers on fMRI data. Here we compared six multivariate classifiers and investigated how the response-amplitude estimate used (beta- or t-value) and different pattern normalizations affect classification performance. The compared classifiers were a pattern-correlation classifier, a k-nearest-neighbors classifier, Fisher's linear discriminant, Gaussian naïve Bayes, and linear and nonlinear (radial-basis-function kernel) support vector machines. We compared these classifiers' accuracy at decoding the category of visual objects from response patterns in human early visual and inferior temporal cortex acquired in an event-related design with BOLD fMRI at 3T using SENSE and isotropic voxels of about 2-mm width. Overall, Fisher's linear discriminant (with an optimal-shrinkage covariance estimator) and the linear support vector machine performed best. The pattern-correlation classifier often performed similarly as those two classifiers. The nonlinear classifiers never performed better and sometimes significantly worse than the linear classifiers, suggesting overfitting. Defining response patterns by t-values (or in error-standard-deviation units) rather than by beta estimates (in % signal change) to define the patterns appeared advantageous. Cross-validation by a leave-one-stimulus-pair-out method gave higher accuracies than a leave-one-run-out method, suggesting that generalization to independent runs (which more safely ensures independence of the test set) is more challenging than generalization to novel stimuli within the same category. Independent selection of fewer more visually responsive voxels tended to yield better decoding performance for all classifiers. Normalizing mean and standard deviation of the response patterns either across stimuli or across voxels had no significant effect on decoding performance. Overall our results suggest that linear decoders based on t-value patterns may perform best in the present scenario of visual object representations measured for about 60min per subject with 3T fMRI.

Noninvasive measurement of cerebral venous oxygenation can serve as a tool for better understanding fMRI signals and for clinical evaluation of brain oxygen homeostasis. In this study a novel technique, T2-Relaxation-Under-Spin-Tagging (TRUST) MRI, is developed to estimate oxygenation in venous vessels. This method uses the spin labeling principle to automatically isolate pure blood signals from which T2 relaxation times are determined using flow-insensitive T2-preparation pulses. The blood T2 is then converted to blood oxygenation using a calibration plot. In vivo experiments gave a baseline venous oxygenation of 64.8 +/- 6.3% in sagittal sinus in healthy volunteers (n = 24). Reproducibility studies demonstrated that the standard deviation across trials was 2.0 +/- 1.1%. The effects of repetition time and inversion time selections were investigated. The TRUST technique was further tested using various physiologic challenges. Hypercapnia induced an increase in venous oxygenation by 13.8 +/- 1.1%. On the other hand, caffeine ingestion resulted in a decrease in oxygenation by 7.0 +/- 1.8%. Contrast agent infusion (Gd-DTPA, 0.1 mmol/kg) reduced venous blood T2 by 11.2 ms. The results of this study show that TRUST MRI is a useful technique for quantitative assessment of blood oxygenation in the brain.

Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential, a procedure for enumerating such cells has to date not been available. We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages. This assay has two key features. The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells. The second is the use of a limiting-dilution experimental design to allow stem cell quantitation. The assay involves transplanting limiting numbers of male "test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation. The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics). Investigation of this assay system has shown that all three potential sources of stem cells (test cells, compromised cells, and the host) can under appropriate circumstances contribute to long-term hematopoietic regeneration, thus establishing both the competitive pressure of hematopoietic stem cells in the cotransplanted compromised population and in the host, and the need to use genetic markers to track the specific contribution of the injected test cells. Analysis of the frequency of CRU in test marrow suspensions that varied widely in their CRU content gave similar values when endpoints of either 5 or 10 weeks posttransplantation were used and when either recipient marrow or thymus was used to identify progeny populations. In addition, repopulation of marrow and thymus was found to be associated in most mice injected with limiting numbers of test cells. These findings are consistent with the conclusion that the assay is highly selective for a very primitive, totipotent, reconstituting hematopoietic stem cell and should therefore be particularly useful in future gene therapy-oriented research as well as for more basic studies of hematopoietic stem cell regulation and differentiation.

The aims of this study were (a) to find a regime of immunization with cholera toxoid in rats which would establish a high density of antitoxin containing cells (ACC) in the lamina propria of the intestine and (b) to determine the origin of the ACC. The best cellular response was achieved by a single i.p. dose of toxoid in FCA followed by an intraintestinal boost 2 wk later. ACC appeared in the thoracic duct lymph 2 days after boosting, reaching a peak of about 200,000 ACC/h at 3--4 days. This was followed by the appearance of large numbers of ACC in the intestine. The i.p. dose of toxoid by itself gave rise to very few ACC in the gut or thoracic duct lymph, but it had clearly primed the gut immune system for a secondary response. Priming was also achieved by the prolonged oral intake of toxoid. The importance of the intestinal route for boosting was shown by the failure of i.p. challenge to give an ACC response in the intestine after i.p. priming and the small response it provoked after oral priming. ACC among thoracic duct lymphocytes (TDL) and in the lamina propria contained predominantly IgA. Two observations indicated that the major source of the lamina propria ACC was from cells that emerged in the thoracic duct lymph after intraintestinal challenge. Firstly, the establishment of a thoracic duct fistula immediately before challenge prevented the appearance of ACC in the intestine. Secondly, many ACC appeared in the intestine of normal rats after the injection of TDL rich in ACC. Although homing of ACC precursors to the gut was not antigen-dependent, the distribution of ACC in the lamina propria was considerably influenced by the site of the intestinal challenge, the density of ACC being greatest at or distal to the site of injection of toxoid into the lumen of the gut.

The JAK/STAT signaling pathway plays important roles in vertebrate development and the regulation of complex cellular processes. Components of the pathway are conserved in Dictyostelium, Caenorhabditis, and Drosophila, yet the complete sequencing and annotation of the D. melanogaster and C. elegans genomes has failed to identify a receptor, raising the possibility that an alternative type of receptor exists for the invertebrate JAK/STAT pathway. Here we show that domeless (dome) codes for a transmembrane protein required for all JAK/STAT functions in the Drosophila embryo. This includes its known requirement for embryonic segmentation and a newly discovered function in trachea specification. The DOME protein has a similar extracellular structure to the vertebrate cytokine class I receptors, although its sequence has greatly diverged. Like many interleukin receptors, DOME has a cytokine binding homology module (CBM) and three extracellular fibronectin-type-III domains (FnIII). Despite its low degree of overall similarity, key amino acids required for signaling in the vertebrate cytokine class I receptors [3] are conserved in the CBM region. DOME is a signal-transducing receptor with most similarities to the IL-6 receptor family, but it also has characteristics found in the IL-3 receptor family. This suggests that the vertebrate families evolved from a single ancestral receptor that also gave rise to dome.

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.

The enteric nervous system (ENS) in mammals forms from neural crest cells during embryogenesis and early postnatal life. Nevertheless, multipotent progenitors of the ENS can be identified in the adult intestine using clonal cultures and in vivo transplantation assays. The identity of these neurogenic precursors in the adult gut and their relationship to the embryonic progenitors of the ENS are currently unknown. Using genetic fate mapping, we here demonstrate that mouse neural crest cells marked by SRY box-containing gene 10 (Sox10) generate the neuronal and glial lineages of enteric ganglia. Most neurons originated from progenitors residing in the gut during mid-gestation. Afterward, enteric neurogenesis was reduced, and it ceased between 1 and 3 months of postnatal life. Sox10-expressing cells present in the myenteric plexus of adult mice expressed glial markers, and we found no evidence that these cells participated in neurogenesis under steady-state conditions. However, they retained neurogenic potential, as they were capable of generating neurons with characteristics of enteric neurons in culture. Furthermore, enteric glia gave rise to neurons in vivo in response to chemical injury to the enteric ganglia. Our results indicate that despite the absence of constitutive neurogenesis in the adult gut, enteric glia maintain limited neurogenic potential, which can be activated by tissue dissociation or injury.

BACKGROUND

In clinical practice, the glomerular filtration rate (GFR) is often estimated from plasma creatinine. Several studies have shown cystatin C (cys C) to be a better parameter for the diagnosis of impaired renal function. No data are available, however, on the performance of cys C in follow-up of patients, compared with creatinine. Also, comparisons of cys C with the Cockcroft and Gault (C&G) formula for estimation of GFR are few.

METHODS

Plasma samples were obtained from 93 consecutive patients seen for GFR determination and from 30 patients with diabetes mellitus type 2, of whom 23 were investigated a second time after 2 years. GFR was determined with [125I]iothalamate. Plasma creatinine was determined enzymatically and the creatinine clearance calculated according to C&G. Cys C was measured with a particle-enhanced immunonephelometric method.

RESULTS

GFR correlated with 1/cys C (r = 0.873) as well as with C&G (r = 0.876). The area under the curve (AUC) of the receiver operating curves (ROCs), a measure of diagnostic accuracy, for cys C (0.931) and C&G (0.938) were equal (P = 0.815) and both better than the creatinine AUC (0.848; P = 0.006). Bland and Altman analysis showed that the simple formula GFR = -4.32 + 80.35 x 1/cys C, derived from our data, gave more accurate (P < 0.0001) and more precise (P = 0.024) GFR estimates than obtained with the C&G formula. The day-to-day variation (biological +analytical) for cys C was small (3.1%, SD 2.51%) in diabetic patients. In the follow-up study in diabetic patients, cys C was the parameter which had the best correlation (r = 0.66) with changes in GFR.

CONCLUSIONS

Cys C shows a high correlation with GFR. With a very simple formula, cys C gives a good estimate of GFR, more accurate and precise than C&G. Because biological variation is low, cys C gives also a good assessment of GFR changes during follow-up. Cys C is the preferred endogenous parameter for GFR.

OBJECTIVE

To assess the reliability of healthcare information on the world wide web and therefore how it may help lay people cope with common health problems.

METHODS

Systematic search by means of two search engines, Yahoo and Excite, of parent oriented web pages relating to home management of feverish children. Reliability of information on the web sites was checked by comparison with published guidelines.

METHODS

Minimum temperature of child that should be considered as fever, optimal sites for measuring temperature, pharmacological and physical treatment of fever, conditions that may warrant a doctor's visit.

RESULTS

41 web pages were retrieved and considered. 28 web pages gave a temperature above which a child is feverish; 26 pages indicated the optimal site for taking temperature, most recommending rectal measurement; 31 of the 34 pages that mentioned drug treatment recommended paracetamol as an antipyretic; 38 pages recommended non-drug measures, most commonly tepid sponging, dressing lightly, and increasing fluid intake; and 36 pages gave some indication of when a doctor should be called. Only four web pages adhered closely to the main recommendations in the guidelines. The largest deviations were in sponging procedures and how to take a child's temperature, whereas there was a general agreement in the use of paracetamol.

CONCLUSIONS

Only a few web sites provided complete and accurate information for this common and widely discussed condition. This suggests an urgent need to check public oriented healthcare information on the internet for accuracy, completeness, and consistency.

Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the bcl-2 protein. However, a recent immunohistochemical survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apoptosis-suppressing oncoprotein at significant levels (Colombel et al., Am. J. Pathol., 143: 390-400, 1993). Additionally, a number of hormone-refractory prostatic adenocarcinomas obtained from hormonally-treated patients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the parental or control-transfected cell lines, LNCaP/bcl-2 cells were highly resistant to a variety of apoptotic stimuli in vitro including serum starvation and 10 nM phorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay. s.c. injections of 10(6) LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of parental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2-transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli in vitro and suggest that such protection correlates with the ability to form hormone-refractory prostate tumors in vivo.

Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.

OBJECTIVE

To investigate the causal role of high-density lipoprotein cholesterol (HDL-C) and triglycerides in coronary heart disease (CHD) using multiple instrumental variables for Mendelian randomization.

RESULTS

We developed weighted allele scores based on single nucleotide polymorphisms (SNPs) with established associations with HDL-C, triglycerides, and low-density lipoprotein cholesterol (LDL-C). For each trait, we constructed two scores. The first was unrestricted, including all independent SNPs associated with the lipid trait identified from a prior meta-analysis (threshold P < 2 × 10(-6)); and the second a restricted score, filtered to remove any SNPs also associated with either of the other two lipid traits at P ≤ 0.01. Mendelian randomization meta-analyses were conducted in 17 studies including 62,199 participants and 12,099 CHD events. Both the unrestricted and restricted allele scores for LDL-C (42 and 19 SNPs, respectively) associated with CHD. For HDL-C, the unrestricted allele score (48 SNPs) was associated with CHD (OR: 0.53; 95% CI: 0.40, 0.70), per 1 mmol/L higher HDL-C, but neither the restricted allele score (19 SNPs; OR: 0.91; 95% CI: 0.42, 1.98) nor the unrestricted HDL-C allele score adjusted for triglycerides, LDL-C, or statin use (OR: 0.81; 95% CI: 0.44, 1.46) showed a robust association. For triglycerides, the unrestricted allele score (67 SNPs) and the restricted allele score (27 SNPs) were both associated with CHD (OR: 1.62; 95% CI: 1.24, 2.11 and 1.61; 95% CI: 1.00, 2.59, respectively) per 1-log unit increment. However, the unrestricted triglyceride score adjusted for HDL-C, LDL-C, and statin use gave an OR for CHD of 1.01 (95% CI: 0.59, 1.75).

CONCLUSIONS

The genetic findings support a causal effect of triglycerides on CHD risk, but a causal role for HDL-C, though possible, remains less certain.

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT-PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of (32)P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys(105) and Lys(127). This segment contained five serines and two threonines. Among these, Ser(106), Ser(123), and Thr(124) were identified as phosphorylated residues; in addition, either one or both of Ser(111) and Ser(112) were phosphorylated. The neighboring residues, Ser(123) and Thr(124), were found in three different phosphorylation states in that either Ser(123) or Thr(124) or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr(701). Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser(639) located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn(653) to Arg(691), at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.