Molecular taxonomy of a soil actinomycete isolate, KCCM10454 showing neuroprotective activity by 16S rRNA and rpoB gene analysis

Epilepsy constitutes a significant public health problem, and even the newest drugs and neurosurgical techniques have proven unable to cure the disease. In order to select a group of isolates which could generate an active compound with neuroprotective or antiepileptic properties, we isolated 517 actinomycete strains from soil samples taken from Jeju Island, in South Korea. We then screened these strains for possible anti-apoptotic effects against serum deprivation-induced hippocampal cell death, using the 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay as an in vitro test. The excitotoxic glutamate analog, kainic acid (KA), was used to induce seizures in experimental mice in our in vivo tests. As a result of this testing, we located one strain which exhibited profound neuroprotective activity. This strain was identified as a Streptomyces species, and exhibited the rifampin-resistant genotype, Asn(AAC)442, according to the results of 16S rRNA and rpoB gene analyses.     

Genetic analysis of the Utah population: a comparison of STR and VNTR loci

Genetic data are reported for nine short tandem repeat (STR) loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820) and six variable number of tandem repeat (VNTR) loci (D2S44, D10S28, D4S139, D1S7, D5S110, and D17S79) in samples of Utah African Americans, European Americans, and Hispanics. Little evidence of departures from Hardy-Weinberg equilibrium or gametic equilibrium was found in these populations. Because of their relatively higher mutation rates, the VNTR loci exhibited higher average heterozygosity and lower FST levels than did the STR loci. Genetic distance analysis showed congruence between the two types of systems, and a genetic distance analysis of the STR data showed that the three Utah populations are genetically similar to the same ethnic groups in other parts of the United States. In addition, this analysis showed that the African American population is the most genetically divergent, with greater similarity between the Hispanic and European American populations. This analysis demonstrates a high degree of consistency for population designations commonly used in forensic analysis.     

Lead dynamics in the forest floor and mineral soil in south-central Ontario

Recent studies have suggested that the residence time of Pb in the forest floor may not be as long as previously thought, and there is concern that the large pulse of atmospheric Pb deposited in the 1960s and early 1970s may move rapidly through mineral soils and eventually contaminate groundwater. In order to assess Pb mobility at a woodland (JMOEC) in south-central Ontario, a stable Pb isotope tracer ²⁰⁷Pb (8 mg m^–2) was added to the forest floor in white pine (Pinus strobus) and sugar maple (Acer saccharum) stands, respectively, and monitored over a 2-year period. Excess ²⁰⁷Pb was rapidly lost from the forest floor. Applying first-order rate coefficients (k) of 0.57 (maple) and 0.32 (pine) obtained from the tracer study, and estimates of Pb deposition in the region, current predicted Pb concentrations in the forest floor are 1.5–3.1 and 2.1–5.8 mg kg^–1 in the maple and pine plots, respectively. These values compare favorably with measured concentrations (corrected for mineral soil contamination) of 3.1–4.3 mg kg^–1 in the maple stand and 2.6–3.6 mg kg^–1 in the pine stand. The response time (1/^k) of Pb in the forest floor at the sugar maple and white pine plots was estimated to be 1.8 and 3.1 years, respectively. The rapid loss of Pb from the forest floor at the JMOEC is much greater than previously reported, and is probably due to the rapid rate of litter turnover that is characteristic of forests with mull-type forest floors. In a survey of 23 forested sites that border the Precambrian Shield in south-central Ontario, Pb concentrations in the forest floor increased exponentially with decreasing soil pH. Lead concentrations in the forest floor at the most acidic survey sites, which exhibited mor-type forest floors, were approximately 10 times higher (80 mg kg^–1) than at the JMOEC, and pollution Pb burdens were up to 25 times greater. Despite the rapid loss of Pb from the forest floor at the JMOEC, the highest pollution Pb concentrations were found in the upper (0–1 cm) mineral soil horizon. Lead concentrations in the upper 30 cm of mineral soil were strongly correlated with organic matter content, indicating that pollution Pb does not move as a pulse down the soil profile, but instead is linked with organic matter distribution, indicating groundwater contamination is unlikely.     

Confirmation of carbadox and olaquindox metabolites in porcine liver using liquid chromatography-electrospray, tandem mass spectrometry

A method is described for the quantitative determination of quinoxaline-2-carboxylic acid (QCA) and methyl-3-quinoxaline-2-carboxylic acid (MQCA), the metabolites that have been designated as the marker residues for the veterinary drugs, carbadox and olaquindox, respectively, in swine tissue. The method is suitable for use as a confirmatory method under EU National Surveillance Schemes. Porcine liver samples were subjected to protease digestion followed by liquid-liquid extraction. Further clean-up was performed by automated solid phase extraction (SPE) and was followed by a final liquid-liquid extraction step. Analysis was performed using a narrow bore column HPLC coupled to electrospray MS/MS, operated in positive ion mode. MS/MS product ions were monitored at m/z 102 and 75 amu for QCA, m/z 145 and 102 amu for MQCA and at m/z 106 and 152 amu for the d(4)-QCA and d(7)-MQCA internal standards, respectively. The method has been validated at 3.0, 10, 50 and 150 microg kg(-1) for both metabolites. The method performance characteristics-the decision limit (CCalpha) and the detection capability (CCbeta) have been determined for QCA at 0.4 and 1.2 microg kg(-1), respectively, and for MQCA at 0.7 and 3.6 microg kg(-1), respectively.     

Insights into the biosynthesis of the benzoquinone ansamycins geldanamycin and herbimycin, obtained by gene sequencing and disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.     

Evaluating rejuvenation of the forehead and brow: an algorithm for selecting the appropriate technique

The traditional reason for performing aesthetic surgery in the forehead and brow area has been to correct brow ptosis. However, there are several other conditions that may be improved by surgery in this area, including frown muscle imbalance, transverse forehead rhytids, and lateral brow laxity. Recently, a better understanding of the relevant anatomy and the evolving therapeutic modalities (including both open and closed techniques) have contributed to a renewed interest in aesthetic surgery in the forehead and brow area. One hundred consecutive patients were studied, each of whom underwent forehead rejuvenation for one of four indications-forehead rhytids, glabellar creases, lateral brow laxity, or brow ptosis. Thirty-eight percent of patients underwent open procedures, 30 percent underwent closed procedures, and 32 percent underwent limited procedures. Complications occurred in 4 percent of patients, including three patients who were dissatisfied with the surgery and one patient who required scar revision. Based on our findings, we formulated an algorithm that integrates the different indications and any concomitant procedures being performed. Our proposed treatment plan is based on this information. The algorithm may be used as a template when assessing a patient and adapting the recommended intervention to the individual patient.     

Temporal perception of sweetness by adults and children using computerized time-intensity measures

There is a general paucity of knowledge of the cognitive and perceptual abilities of children to successfully undertake chemosensory-related tasks. An example is that there are no reports of temporal perception by children in time-intensity tasks, or how their responses in these tasks compare with those of adults. The latter paradigm has the potential to reveal differences that may occur during a normal eating or drinking episode that cannot be detected with single response measures. To address this shortcoming, the present study uses a computerized time-intensity method to compare the responses of adults and 8- to 9-year-olds in several measures of sweetness with three different types of stimuli. The results show that the children gave higher estimates than adults of the maximum sweetness of sucrose in water, orange drink and custard and recorded shorter sweetness durations with orange drink and custard. Both age groups, however, responded similarly to changes in concentration and the volume of stimuli with all three sensory measures. Overall, the consistency of the data from the children and the variability, which was similar to that of the adults, indicate that the tasks involved in the time-intensity paradigm were within the cognitive ability of the children. Nevertheless, further studies are needed to determine the basis of the differences found.     

Mechanisms linking under-nutrition and ovarian function in beef heifers

Prolonged reduction in energy intake in beef heifers has been reported to suppress ovulation but the mechanisms involved are poorly understood. The objective of this study was to examine whether changes in the pattern of LH secretion following each of three different tests predicted the functional state of the hypothalamo-pituitary-ovarian (H-P-O) axis. Test 1 examined the ratio of LH secretion during the 1h before and 2h after naloxone (NAL) administration. The other two tests assessed the LH surge following an exogenous oestradiol positive feedback signal (Test 2) or exogenous progesterone priming (Test 3). In phases 1 and 3, each of 8 weeks duration, the heifers were fed 100% of their maintenance energy requirements. In phase 2, of 9 weeks duration, they were fed 50% of their maintenance energy requirements. Oestrus was induced in all heifers by PG administration at the start of the experiment.Heifers were administered a naloxone challenge of 50, 100, 200 or 400mg naloxone hydrochloride i.v. (one dose per heifer) during the mid-luteal period of phase 1 and all four naloxone treated heifers received 400mg naloxone hydrochloride at the end of phases 2 and 3. Doses of 10, 20 or 40 mg oestradiol benzoate (EB) i.m. were each administered to two of the remaining heifers during the mid-luteal period of phase 1. One heifer on each dose of oestradiol benzoate in phase 1 had the same dose administered at the end of phases 2 and 3. The progesterone challenge was administered to three heifers by insertion of a PRID for 12 days starting in the middle of phase 2.In Test 1, the ratio of LH secretion before and after naloxone administration in phase 1 was 1:1 (50mg), 1:4 (100mg), 1:4 (200mg) and 1:9 (400mg) (50mg versus 100mg and 100mg versus 200mg doses, P<0.05); 50mg versus 400mg doses, P<0.001). In phase 2, this ratio was 1:1 and there was no response to 400mg dose of naloxone in any of the four heifers. In phase 3, the ratio depended on the ovarian activity in the heifer and ranged from 1:1 to 1:4 (P<0.05). In Test 3, a positive oestradiol feedback signal was detected in cyclic heifers in phases 1-3 but not in the acyclic heifer in phase 2. Heifers challenge with exogenous progesterone did not have oestradiol or LH values above threshold levels.We conclude that all three tests successfully predicted the functional state of the hypothalamo-pituitary-ovarian axis. In nutritionally undernourished beef heifers onset of ovarian acyclicity is either preceded or accompanied by the loss of a positive feedback signal (Test 2) and progesterone priming ability (Test 3), and that a plasma LH ratio of > or =1:2 following naloxone challenge (Test 1) is a sign of recovery of the functional state of the hypothalamo-pituitary-ovarian axis.     

Narrowing the critical region within 11q24-qter for hypoplastic left heart and identification of a candidate gene, JAM3, expressed during cardiogenesis

Hypoplastic left heart is a severe human congenital heart defect characterized by left ventricular hypoplasiawith aortic and mitral valve atresia. A genetic etiology is indicated by an association of the hypoplastic left heart phenotype with terminal 11q deletions that span approximately 20 Mb (distal to FRA11B in 11q23). Here we define the breakpoints in four patients with heart defects in association with distal 11q monosomy and refine the critical region to an approximately 9-Mb region distal to D11S1351. Within this critical region we have identified JAM3, a member of the junction adhesion molecule family, as a strong candidate gene for the cardiac phenotype on the basis that it is expressed during human cardiogenesis in the structures principally affected in hypoplastic left heart.