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PubMed1991

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.

Base Sequence↗
PubMedDate not supplied

Circumcision--an update.

Circumcision will likely continue to be a frequently performed procedure during the neonatal period. Rather than allowing it to be an emotion-laden issue, clinicians should keep abreast of ongoing developments in the field and be able to present thorough, objective counseling. Informed parental refusal requires no less.

Anesthesia, Local↗
PubMed1988

Affinity of microorganisms of the genus ureaplasma to the reproductive organs of cattle.

The purpose of this work was to define more precisely the role of Ureaplasma organisms in the aetiology of granular vulvovaginitis and balanoposthitis (GVVBP) of cattle. To contribute to this question the frequency and degree of infection with Ureaplasmas in two main groups of cattle was taken into account: (a) in cattle with symptoms of the mentioned disease, (b) in cattle without clinical symptoms. The samples of semen from 301 sires with symptoms of GVVBP and from 43 healthy sires as also vaginal mucus swabs from 96 cows with GVVBP and from 40 cows mated by the sire infected with Ureaplasma organisms and from 50 cows inseminated with semen which contained Ureaplasma organisms were taken for bacteriological examinations. The control group in relation to the above mentioned cows constituted of 22 heifers free from symptoms of GVVBP and neither inseminated nor mated naturally. It has been shown that on an average 78.1% of sires with pathological changes in the mucosa of the penis or prepuce and only 25.6% of healthy sires were infected with Ureaplasma organisms. The concentration of Ureaplasma organisms was also significantly higher in material obtained from sires with symptoms of the disease than in that from healthy animals. Ureaplasma organisms were demonstrated more frequently (72.7%) in cows with GVVBP than in cows without these symptoms (13.3%). Similarly, as in the material obtained from sires, in the material taken from cows with symptoms of the disease the concentration of Ureaplasma organisms was significantly higher than that in the material originating from the healthy cows. The obtained findings may indicate that Ureaplasma organisms play a role in the aetiology of GVVBP.

Animals↗
PubMed1993

Development of the tuberoinfundibular system in rats: birthdates of the tyrosine hydroxylase-immunopositive neurons.

The present study has attempted to determine the birthdates of the tyrosine hydroxylase (TH)-immunopositive neurons in the arcuate nucleus (AN) of rats, thus evaluating the time of the last mitotic divisions of their cell precursors. For this aim, 'long-survival' [3H]thymidine autoradiography was used in combination with immunocytochemistry of TH, the first enzyme of catecholamine synthesis. According to our data, some TH-immunopositive neurons in the AN were born as early as the 13th fetal day. At day 14, the number of double-labeled neurons almost tripled while reaching a maximum at day 15 of the intrauterine development. The fraction of the double-labeled neurons dropped on the following day, and practically disappeared by fetal day 17, showing cessation of the production of the TH-immunopositive neurons. The birth of the TH-immunopositive neurons is considered as the onset of the development of the tuberoinfundibular dopaminergic system.

Animals↗
PubMed1995

Thermostabilization of live virus vaccines by heavy water (D2O).

Eradication of poliomyelitis is based on the mass administration of oral poliovirus vaccine (OPV). Delivery of effective vaccines in the developing world, especially in tropical areas, is compromised when refrigeration cannot be assured. The OPV, prepared with three live attenuated polioviruses (Sabin strains, serotypes 1, 2 and 3), is considered to be the most thermolabile of vaccines in the World Health Organization's Expanded programme on Immunization. To be effective, the initial concentration (potency of each of the three component serotypes, measured in tissue culture infective doses, should not decrease by more than 0.5 log10 before vaccine delivery. High concentration (1 M) of MgCl2 is currently used as stabilizer for OPV. The stabilizing effect of D2O was tested here on OPV strains. By diluting the viral suspension with D2O-based salt and buffer solutions, in a manner similar to that involved in OPV production an 87% concentration of D2O in the final viral preparation was achieved. In severe conditions of testing (incubation for 3 days at 45 degrees C), the Sabin 3 virus lost an average of 2.7 log10 potency in the presence of 87% D2 as compared to 3.0 log10 in H2O-based 1 M MgCl2, and to 5.7 log10 in the H2O-based control solutions. When tested in a combined 87% D2O and 1 M MgCl2 treatment, the Sabin 3 virus lost only 1.3 log10 potency after 3 days at 45 degrees C. Similar thermostabilizing effects were obtained for Sabin 1 and Sabin 2 strains, but the level of stabilization was slightly lower. Tested in standard conditions at 37 degrees C for 7 days, the infectivity of the three D2O MgCl2-treated OPV strains remained in the limit of requirements ( < or = 0.5 log10). The stabilizing effect of D2O was also demonstrated on yellow fever 17D vaccine virus strain.

Cell Line↗
PubMed2006

Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits.

Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.

Acetylcholine↗
PubMed2004

FADD-dependent apoptosis induction in Jurkat leukemia T-cells by the resveratrol analogue, 3,4,5-trihydroxy-trans-stilbene.

The plant-produced compound, resveratrol (3,5,4'-trihydroxy-trans-stilbene, 3,4,5-THS), induces apoptosis in various human leukemia cell types in vitro, and thus appears to be a promising anti-leukemia agent. In this study, we observed that treatment of resveratrol-resistant Jurkat cells with the resveratrol analogue, 3,4,5-trihydroxy-trans-stilbene (3,4,5-THS), rapidly induced extensive apoptosis, indicating that the apoptotic activity of the analogue differed from that of the parental compound resveratrol. Indeed, we found that treatment of Jurkat cells with 3,4,5-THS, unlike treatment with resveratrol, induced activation of caspase-8 and apoptosis by a Fas-associated death domain (FADD) protein-dependent mechanism without involving the known death ligands CD95 ligand (CD95L), tumor necrosis factor alpha (TNFalpha) and TNF-related apoptosis-inducing ligand (TRAIL). Therefore, 3,4,5-THS induced activation of a FADD-dependent apoptotic mechanism that was unresponsive to the parental compound resveratrol. Therefore, the ability of 3,4,5-THS, but not resveratrol, to induce apoptosis demonstrates a structure-associated apoptotic activity of the resveratrol analogue.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Down-regulation of estrogen receptor-alpha in MCF-7 human breast cancer cells after proteasome inhibition.

The eukaryotic proteasome is a 26S ATP-dependent proteolytic complex, which possesses chymotrypsin-like, trypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities, which enable the proteasome to degrade all short-lived and many long-lived proteins, and consequently regulate a myriad of activities in cells. In this study, we observed that inhibition of the proteasome, and more specifically, inhibition of the chymotrypsin-like activity of the proteasome, in MCF-7 human breast cancer cells resulted in selective down-regulation of the nuclear estrogen receptor-alpha (ERalpha). Our data indicated that estrogen had no effect, whereas the ERalpha antagonist, tamoxifen, reduced the amount of ERalpha that could be subjected to down-regulation after proteasome inhibition. Furthermore, our data demonstrated that protein synthesis was required for the down-regulation of ERalpha to occur. Collectively, these data indicate the existence of a proteasome-dependent mechanism that is utilized by MCF-7 cells to maintain a steady-state level of ERalpha.

Breast Neoplasms↗
PubMed2006

Proteasome-independent down-regulation of estrogen receptor-alpha (ERalpha) in breast cancer cells treated with 4,4'-dihydroxy-trans-stilbene.

Treatment of cells with estrogens and several pure ERalpha antagonists rapidly induces down-regulation of the alpha-type estrogen receptor (ERalpha) in the nucleus by mechanisms that are sensitive to the proteasome inhibitors, MG132 and clasto-lactacystin-beta-lactone. Hence, it is believed that these ER ligands induce down-regulation of ERalpha by proteasome-dependent mechanisms, which serve to control both the amount of transcriptional activity and the level of ligand-bound ERalpha in cells. In this study, we observed that treatment of cultured MCF-7 and T47D human breast cancer cells with the low affinity ER ligand, 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), inhibited the transcriptional activity of ERalpha and induced slow and gradual decrease in the amount of ERalpha protein (henceforth referred to as down-regulation of ERalpha). The 4,4'-DHS-induced down-regulation of ERalpha in MCF-7 cells involved a mechanism that was insensitive to the two most specific proteasome inhibitors, clasto-lactacystin-beta-lactone and epoxomycin, but sensitive to MG132 at concentrations exceeding that required for maximal inhibition of the proteasome in MCF-7 cells. Therefore, 4,4'-DHS appears to induce down-regulation of ERalpha by a proteasome-independent mechanism. Here, we present data to show that both 4-OH and 4'-OH are critical for the ability of 4,4'-DHS to induce down-regulation of ERalpha and suggest that 4,4'-DHS provides a useful scaffold for development of novel ERalpha antagonists.

Base Sequence↗
PubMedDate not supplied

Parallel computation of simple arithmetic using peptide-antibody interactions.

We propose a theoretical model for representing and manipulating binary numbers using peptide-antibody interactions. In particular, we present models to solve simple binary arithmetical problems like addition and subtraction. As the interactions can take place in parallel we show that the number of steps is independent of the size (bits) of the numbers.

Algorithms↗
PubMed2005

Dynamic contrast enhanced magnetic resonance imaging and magnetic resonance spectroscopy in diabetic mastopathy.

Diabetic mastopathy is a rare, benign clinico-pathological entity strongly associated with type I diabetes. X-ray mammography and ultrasonography are inadequate to distinguish this lesion from malignancy, leading to unnecessary excision biopsies. Dynamic contrast enhanced MRI and MR spectroscopy, powerful tools in the investigation of breast disease, can help solve this problem.

Adult↗
PubMed2006

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: role of water activity.

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)-iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)-nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A20, G20, C20 and T20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.

Adsorption↗
PubMed2005

16-Detector multislice CT in the detection of stress fractures: a comparison with skeletal scintigraphy.

AIMS: To test the hypothesis that the improved resolution afforded by 16-detector computed tomography (CT) would translate to better stress fracture detection when compared with skeletal scintigraphy. MATERIALS AND METHODS: Thirty-three cases of suspected stress fractures in 26 patients were investigated using skeletal scintigraphy and 16-detector CT performed on the same day. Planar images of the lower limbs were taken 3h post-injection of 400MBq (99m)Tc-methylene diphosphonate ((99m)Tc-MDP). (99m)Tc-MDP uptake was quantified at suspected fracture sites. CT was performed using a 16-detector multisection machine employing 0.75mm detectors and images reconstructed in 0.5mm increments. Examinations were reported independently and discordant results were compared at follow-up. RESULTS: At initial reporting scintigraphy identified fractures in 13 of the 33 cases and CT identified four of the 33. In one case, on review of the CT images, a fracture was present in the distal fibula that was not initially identified. This resulted in eight scintigraphic-positive CT-negative discordant cases. The (99m)Tc-MDP uptake was significantly lower in the discordant fracture group compared with the concordant group (p<0.01). CONCLUSIONS: Despite technological advances in CT, scintigraphy appeared to detect more stress fractures. As such, multidetector CT should not be used as a routine initial investigation in stress fracture detection. The potential use of (99m)Tc-MDP quantification at fracture sites is of interest and may be worth further investigation.

Adolescent↗
PubMed2005

Ultrasonography, computed tomography and magnetic resonance imaging in the assessment of pelvic pathology.

OBJECTIVE: Ultrasound (US) is the primary imaging modality in the investigation of pelvic pathology in women however it can be very inaccurate. MRI and CT provide a more detailed pelvic examination and hence we compared their accuracies with that of ultrasound to find out if these two modalities should be used more often. PATIENTS AND METHODS: 136 women who had MRI examination of the pelvis for investigation of probable pelvic pathology were studied. Hundred and twenty-five of these women had an initial ultrasound scan and 23 had an initial CT. Diagnostic accuracy was assessed against histopathology or clinical follow-up. RESULTS: Histopathology was available in 127/136 women. Overall 36% of the lesions were malignant. The overall accuracy of MRI, US and CT were 97%, 77% and 87%, respectively. MRI confidently identified the tissue of origin in 94% compared to only 66% for US. There was a significant difference in accuracy between MRI and US in diagnosing adnexal and uterine pathology. MRI was better than CT and US in diagnosing peritoneal metastases whereas CT was superior in diagnosing omental infiltration. CONCLUSION: We suggest that all women with a pelvic abnormality identified on US or in whom there is a strong clinical suspicion of disease should undergo MR pelvic imaging in preference to CT because of its better soft tissue resolution and multi-planar capability.

Adult↗
PubMed2025

Rethinking the pathogenicity of intragenic DMD duplications detected by carrier screening: High prevalence of nontandem duplications revealed by long-read sequencing.

PURPOSE: The pathogenicity of intragenic duplications depends on their structural configuration. Tandem duplications often disrupt reading frames and cause gene loss of function, whereas interspersed (nontandem) duplications are largely benign. When the configuration cannot be determined, current guidelines presume a tandem structure, leading to some laboratories automatically classifying such variants as likely pathogenic or pathogenic. This study evaluates the validity of this presumption for DMD, in patients with and without clinical indications of dystrophinopathy. METHODS: We performed high-coverage long-read genome sequencing on 15 patients with intragenic DMD duplications. A total of 4 patients had clinically indicated dystrophinopathy testing, whereas in the remaining 11 patients, the duplications were detected without clear indications of dystrophinopathy (eg, through carrier screening). RESULTS: All 4 patients with clinical indications had tandem duplications. In contrast, 64% (7/11) of the cases without such indications had interspersed duplications, with 4 subsequently reclassified as likely benign, 2 (likely) pathogenic, and 1 uncertain. These duplications were often complex, involving coduplications or codeletions with other regions. CONCLUSION: Our findings challenge the presumption that intragenic DMD duplications are predominantly in tandem. This highlights the need for a cautious variant interpretation approach, particularly in carrier screening and other settings in which variants are identified without indications of dystrophinopathy.

Humans↗