Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.     

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.     

OKN, perceptual and VEP direction biases in strabismus

The present study quantified nasalward/temporalward biases in monocular optokinetic nystagmus (MOKN) and perceived velocity in patients with either early onset esotropia, late onset esotropia and in normals. MOKN was measured with low spatial frequency, small-field gratings drifting at 9.4 degrees/s. MOKN bias was quantified as the ratio of nasalward slow-phase velocity divided by the sum of temporalward and nasalward slow-phase velocities (N/(N + T)). Observers also rated the perceived velocity of gratings moving in nasalward and temporalward directions (3 or 9.4 degrees/s) using a two interval forced choice task. MOKN and perceived velocity biases were correlated negatively in both early onset and late onset groups in the perceptual task--nasalward moving targets were rated as slower than temporalward targets, but in the MOKN task, slow-phase gain was higher for nasalward than for temporalward targets. Oscillatory-motion, visual evoked potentials (VEPs), were recorded in response to 1 c/deg gratings undergoing apparent motion at 10 Hz in a subset of the observers. VEP direction biases were quantified by calculating the ratio of first harmonic response amplitudes to the sum of first and second harmonic amplitudes. Significant correlations were found between the direction biases obtained on all three measures. Perceived velocity and MOKN bias measures were also correlated negatively. Patients with early onset esotropia (infantile esotropia) had larger biases than late onset esotropes or normals on each measure and the biases were more frequently bilateral in the early onset patients. The pattern of result is consistent with early critical periods for the mechanism(s) underlying MOKN, perceived velocity and cortical responsiveness. A single site model for all three asymmetries is unlikely, at least in simple form, because of the negative correlation between MOKN and perceived velocity biases and because of the differences in relative magnitude between the perceptual and MOKN biases.     

Dental implant outcomes in postmenopausal women undergoing hormone replacement

To determine if hormone supplementation reduces the risk of failure for osseointegration of dental implants in postmenopausal women, the treatment outcomes of 116 women older than age 50, treated with 450 endosseous dental implants, were analyzed in this retrospective study. The findings indicated that hormone replacement therapy may not be linked with improved outcomes of endosseous dental implant treatment in postmenopausal women. Smoking, however, appears to significantly increase the implant failure rate in the group observed in this study.     

The surgical treatment of a large postoperative ventral hernia in patients over 60

Breech presentation occurs in 3-5% of deliveries and can be managed by either a trial of vaginal breech delivery (TOVBD), external cephalic version (ECV) or Caesarean section. A postal questionnaire was completed by 82% of Scottish consultant obstetricians and revealed wide variations in practice. Eighteen percent never offered ECV. Among those who did consensus was lacking on some contraindications. One-quarter sometimes performed ECV before 37 weeks gestations despite the possibility of spontaneous version. Only 70% restricted ECV to one or more designated operators thereby maintaining levels of expertise. Variations were demonstrated in the use of tocolytics, and pre and post procedure investigations. Following failed ECV 28% considered a repeat attempt and 56% a TOVBD. TOVBD was not offered as first line management by one-fifth of respondents. Those who did varied in the pre-procedure investigations performed. Guidelines are required to ensure safe, consistent practice and avoid unnecessary Caesarean sections.     

UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase

We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7-ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4',6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified.     

Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells

Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.