Effects of stem cell factor on in vitro growth of buffalo oocytes.

Stem cell factor (SCF) plays important roles in primordial follicle activation, oocyte growth and survival, granulosa cell proliferation, theca cell recruitment, and ovarian steroidogenesis. The aim of this study was to investigate the effect of SCF on in vitro growth of buffalo oocytes. Oocyte-granulosa cell complexes (OGCs) were dissected from early antral follicles of slaughtered buffalo ovaries and cultured for 6 days in media supplemented with 0, 50 or 100 ng/mL SCF. In vitro grown oocytes were further cultured for in vitro maturation for 24 h. The results showed that SCF significantly (P < 0.05) increased oocyte diameter in vitro. The percentages of surviving oocytes were 60, 81 and 92 in 0, 50 and 100 ng/mL SCF supplemented group, respectively. SCF promoted formation of antrum-like structures in culture. The results also showed that SCF enhanced the maturation of in vitro grown buffalo oocytes. Here, 14% in vitro grown oocytes reached metaphase II (MII) stage in 50 ng/mL SCF supplemented group, whereas the percentage was increased to 26% in 100 ng/mL SCF treated group. These results show that SCF supports the growth, viability and nuclear maturation of buffalo oocytes in vitro.

First trimester fetal heart rate as a predictor of newborn sex.

OBJECTIVE: To predict the sex of newborns using first trimester fetal heart rate (FHR). METHODS: This was a retrospective review of medical records and ultrasounds performed between 8 and 13 weeks of gestation. Continuous variables were compared using Student's t-tests while categorical variables were compared using Chi-square test. RESULTS: We found no significant differences between 332 (50.7%) female and 323 (49.3%) male FHRs during the first trimester. The mean FHR for female fetuses was 167.0 ± 9.1 bpm and for male fetuses 167.3 ± 10.1 bpm (p = 0.62). There was no significant difference in crown rump length between female and male fetuses (4.01 ± 1.7 versus 3.98 ± 1.7 cm; p = 0.78) or in gestational age at birth (38.01 ± 2.1 versus 38.08 ± 2.1 weeks; p = 0.67). The males were significantly heavier than females (3305.3 ± 568.3 versus 3127.5 ± 579.8 g; p < 0.0001) but there were no differences in the proportion of small for gestational age (SGA), average for gestational age (AGA) and large for gestational age (LGA) infants. CONCLUSIONS: We found no significant difference between the female and male FHR during the first trimester in contrast to the prevailing lay view of females having a faster FHR. The only statistically significant difference was that males weighed more than female newborns.

CYP2D6 Metabolism in Frail Elderly Compared to Non-Frail Elderly: A Pilot Feasibility Study.

BACKGROUND: Frailty is a clinical phenotype that is associated with adverse health outcomes. Since frail patients may be more prone for adverse drug events and about 15-20 % of commonly prescribed drugs are metabolized by CYP2D6, we hypothesized that CYP2D6 metabolism is decreased in frail patients compared with healthy subjects. METHODS: The (13)C-dextromethorphan breath test (DM-BT) was used to determine CYP2D6 phenotype using (13)C-dextromethorphan ((13)C-DM) as a probe. Eleven frail and 22 non-frail (according to the Fried criteria) subjects aged 70-85 years were phenotyped for CYP2D6. RESULTS: Despite inequalities in CYP2D6 genotype between frail and non-frail subjects, the CYP2D6 gene activity score was equally distributed between the two groups (1.33 ± 0.50 vs. 1.28 ± 0.752). In male patients, no difference in total and free serum testosterone levels was observed between frail and non-frail men. Serum dehydroepiandrostenedione sulfate (DHEAS) levels were lower in frail subjects (1.56 µmol/L) compared with non-frail subjects (2.36 µmol/L), but the difference was not significant (p = 0.15). Body mass index was significantly correlated to CYP2D6 phenotype, whereas frailty score and individual parameters of frailty, Karnofsky score, and activities of daily living score were not significantly correlated to CYP2D6 phenotype. Although there was no difference in CYP2D6 phenotype observed between frail mean ± standard deviation (mean ± SD) area under the curve for delta over baseline values (0-2 h) (AUCDOB2h) 319 ± 169 ‰ min] and non-frail subjects (mean ± SD AUCDOB2h 298 ± 159 ‰ min), the present sample size is considered too small to draw any firm conclusions regarding a potential phenoconversion of CYP2D6 in frail elderly as compared with healthy subjects. CONCLUSION: Frail and non-frail subjects did not differ in CYP2D6 phenotype, taking into account that the precalculated sample size was not achieved. Further studies with more patients are needed in order to adequately understand a possible correlation.

Further characterization of a ¹³C-dextromethorphan breath test for CYP2D6 phenotyping in breast cancer patients on tamoxifen therapy.

In a previous study, we found that the CYP2D6 phenotype determined by (13)C-dextromethorphan breath test (DM-BT) might be used to predict tamoxifen treatment outcome in breast cancer patients in the adjuvant setting. However, large variation in the delta-over-baseline (DOB) values was observed in the extensive metabolizer predicted phenotype group based on single point measures. In the present work we aimed to analyze the variability of phenotype results and determine reproducibility to further characterize the clinical utility of DM-BT by introducing multiple breath sampling instead of single breath sampling and by administration of a fixed dose of (13)C-DM.

Ultrasonographic evaluation of breast mass.

Breast cancer is the second most common cause of cancer-related mortality among women. The aim of the present study is to investigate the diagnostic accuracy of ultrasonography in correlation with histopathology in suspected patients of breast mass. In this cross-sectional study, ultrasonography and histopathology was done on 50 clinically suspected patients of breast mass in the Department of Radiology and Imaging Mymensingh Medical College Hospital, Mymensingh from 1st April 2008 to 30th March 2009 for the period of one year. Women of all ages were included in the study. Findings of USG and histopathology were correlated. In diagnosis of malignant mass by USG, 32(64%) of cases were diagnosed as malignant and 18(36%) cases as other than malignant. Here 31(86.1%) of sonographically diagnosed malignant lesions were also proved as malignant lesion by histopathologically and 1(7.1%) other than malignant. Out of 18 sonographically diagnosed cases of other than malignant lesions 13(92.9%) were proved histopathologically and 5(13.9%) did not match with sonographic findings as other than normal. USG, in diagnosis of malignant lesion, sensitivity was 86.1%, specificity 92.9%, positive predictive value (PPV) (96.6%), negative predictive value 72.2% and accuracy was 88.0% and comparable to other study. In the conclusion this study permits to conclude that ultrasonography has significant sensitivity, specificity, accuracy, positive predictive value and negative predictive value in the diagnosis of both benign and malignant breast mass.

The use of the 13C-dextromethorphan breath test for phenotyping CYP2D6 in breast cancer patients using tamoxifen: association with CYP2D6 genotype and serum endoxifen levels.

PURPOSE: Adjuvant therapy with tamoxifen significantly reduces breast cancer recurrence and mortality in estrogen receptor positive disease. CYP2D6 is the main enzyme involved in the activation of the prodrug tamoxifen into the anti-estrogen endoxifen. Endoxifen is thought to be a main determinant for clinical efficacy in breast cancer patients using tamoxifen. As the large interindividual variation in endoxifen levels is only partly explained by CYP2D6 genotype, we explored the use of the (13)C-dextromethorphan breath test (DM-BT) for phenotyping CYP2D6 and to predict serum steady-state endoxifen levels as a marker for clinical outcome in breast cancer patients using tamoxifen. METHODS: In 65 patients with early breast cancer using tamoxifen, CYP2D6 phenotype was assessed by DM-BT. CYP2D6 genotype using Amplichip and serum steady-state levels of endoxifen were determined. Genotype was translated into the gene activity score and into ultrarapid, extensive, heterozygous extensive, intermediate or poor metabolizer CYP2D6 predicted phenotype. RESULTS: CYP2D6 phenotype determined by the DM-BT explained variation in serum steady-state endoxifen levels for 47.5% (R(2) = 0.475, p < 0.001). Positive and negative predictive values for a recently suggested threshold serum level of endoxifen (5.97 ng/mL) for breast cancer recurrence rate were 100 and 90%, respectively, for both CYP2D6 phenotype by DM-BT (delta-over-baseline at t = 50 min (DOB(50)) values of 0.7-0.9) and genotype (CYP2D6 gene activity score of 1.0). CONCLUSION: DM-BT might be, along with CYP2D6 genotyping, of value in selection of individualized endocrine therapy in patients with early breast cancer, especially when concomitant use of CYP2D6 inhibiting medication alters the phenotype.

Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma.

A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C(18) column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 microg/ml for HPLC and from 0.25 to 20 microg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 microg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29-30 2'-O-Me nucleotides, 1-2 other 2'-modified nucleotides at positions U4 and U7, and a 3'-3'-linked nucleotide "cap." Eight 2'-modified uridine residues were introduced at positions U4 and U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2'-NH2 substitutions at U4 and U7, or 2'-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only approximately 0.1 min. The addition of a 3'-3'-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to > 260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000-80,000-fold compared to the all-RNA parent ribozyme.

The evaluation of skin prick test in house dust mite allergy in Calcutta, India.

Immediate type allergy towards house dust and house dust mites was measured in 188 dust-sensitive asthmatic patients in and around Calcutta by using the skin prick test. Of the 131 positive patients, 82% reacted to Dermatophagoides mites, 80% to D. farinae, 46% to D. pteronyssinus, and 43% to both species of mites. Sixty-two per cent of the positive patients showed strong skin reaction to D. farinae as compared to 32% to D. pteronyssinus. Skin reaction (positive/strong) was highest in D. farinae as compared to other allergens tested in the present study. Skin test results were also analysed in relation to patients' age, sex and duration of disease.

Role of cockroaches in allergy to house dust in Calcutta, India.

Prick skin tests on 200 patients with a history of sensitivity to dust were done against house dust, cockroach, D. pteronyssinus, and D. farinae antigenic extracts. The frequency of positive reaction was 35% to cockroach, 59% to house dust, 84.7% to D. farinae, and 54% to D. pteronyssinus. Distinctively different prevalence of the three antigens, cockroach, house dust, and mites suggest that cockroach antigen is a specific antigen independent from house dust and mite antigens.

Clinical significance of IgE in bronchial asthma.

Total mean serum IgE levels of 63 patients (aged 16-49 years) suffering from bronchial asthma and 64 control subjects (aged 19-40 years) were estimated by RIA method. Mean serum IgE level of patients and control subjects was 1132 +/- 643 units/ml and 43 +/- 26 units/ml respectively. The difference between the two values was statistically significant (p less than 0.001). Four per cent of the patients had serum IgE levels within normal limits. No significant correlation was found between mean serum IgE level of the patients and the duration of the disease. Differences in mean serum IgE level of male and female patients and control subjects was not statistically significant. The mean serum IgE level of patients in the age group of 21-40 years was highest (F = 1.33, p less than 0.05). Significantly elevated levels of serum IgE were observed in patients with both personal and family history of atopic disease in comparison to patients with only personal or family history only (p less than 0.05). As such the estimated raised level of IgE was significant in view of the severity of atopic asthma.

Pentylenetetrazole-induced changes in cerebral energy metabolism in Tupaia glis.

The convulsant pentylenetetrazole was administered to the lower primate, the tree shrew. Shortly after the onset of seizures, the animals were killed using a microwave device at 25 Kw and 915 MHz. The energy metabolites glycogen, glucose, ATP, and phosphocreatine were measured in five layers of the cerebral cortex and three layers of the cerebellum. Results showed that, as compared with controls, seizing animals had decreased energy metabolites selective to certain layers. Glucose was decreased in all cortical layers, but only in the granular layer of the cerebellum. Phosphocreatine was decreased in the outer small pyramidal layer and the polymorphous layer of the cortex but was unchanged in the cerebellum. ATP was decreased only in the outer small polymorphous layer of the cortex. These changes are consistent with the concept that selective changes may occur during seizures and that these changes are localized to layers that contain pyramidal cells. Examination of whole cortex may mask more subtle regional changes.

Mecamylamine, but not atropine, antagonizes nicotine-induced hyperglycemia and potentiation of hypnosis produced by pentobarbital in mice.

An anesthetic dose of 50 mg/kg (i.p.) sodium pentobarbital caused a 61% increase in blood glucose levels in mice. Nicotine (2.5 mg/kg) given intraperitoneally 15 min prior to the sodium pentobarbital treatment further increased hyperglycemia by 29% over pentobarbital alone and 90% higher than the control. Mecamylamine (0.5 mg/kg) given intraperitoneally 15 min prior to nicotine resulted in blood glucose concentrations near the control value. Atropine (2 mg/kg) administered intraperitoneally did not prevent the hyperglycemia induced by nicotine and pentobarbital. No significant correlation was observed between the sleep time and the blood glucose of the unconscious or awake mice. However, a significant correlation was observed between the blood glucose concentration of the sleeping and awake mice. Blood glucose levels were always higher when the neuronal activity was depressed and were lower when the neuronal activity was increased.

Cerebral acetylcholine and energy metabolism changes in acute ammonia intoxication in the lower primate Tupaia glis.

Ammonia levels are elevated in many patients with hepatic encephalopathy. This observation, coupled with animal studies showing an encephalogenic role for ammonia, has led to the concept that ammonia is an important toxin in the production of neurologic symptoms. Studies in rodents have shown that ammonia alters cerebral energy metabolism in the reticular formation, an area important in the modulation of consciousness. Our study was undertaken to extend these observations to the lower primate Tupaia glis, the tree shrew. The energy metabolites glucose, glycogen, lactate, adenosine triphosphate, and phosphocreatine were measured in the reticular formation by microanalytic techniques and enzymatic cycling. Acetylcholine was measured in brain regions by gas chromatography. Acetylcholine levels were increased significantly only in the medulla-pons and diencephalon in the coma stage. The energy metabolites glucose, glycogen, and phosphocreatine were decreased in reticular formation cells during the coma, whereas lactate was increased. During the precoma, glycogen and phosphocreatine were decreased. It appears, therefore, that the tree shrew has a metabolic response to ammonia similar to that of mice. A lowering of energy metabolism in the area of brain-regulating consciousness may act to place the animal in a coma. This coma in turn acts to decrease overall metabolic demand, which allows the animal an opportunity to conserve its threatened energy reserves.

Evaluation of microwave irradiation technique to quantitate acetylcholine in the diaphragm.

This report evaluates rapid enzyme inactivation prior to acetylcholine quantitation in the diaphragm. Methods of sacrifice were decapitation and microwave irradiation. Six-hundred millisecond exposure to microwave irradiation was sufficient to raise diaphragmatic temperature to 91 degrees C and inactivated 90% of the acetylcholinesterase enzyme. The acetylcholine content of the diaphragm was found to be 3.0 nmol/g, independent of the method of sacrifice. The postmortem changes observed in brain acetylcholine content following decapitation did not occur in the diaphragm.

Nicotine potentiates sodium pentobarbital but not ethanol induced sleep.

Nicotine potentiates, in a dose dependent manner, the sleep time induced by sodium pentobarbital but not by ethanol. Mecamylamine, a nicotinic receptor antagonist, blocked the nicotine induced increase in sleep time. Atropine itself reduced sleep time but did not change the nicotine effect. It is hypothesized that the central and the peripheral nicotinic receptors play an important role in potentiating sodium pentobarbital induced sleep time.