Actin isoform utilization during differentiation and remodeling of BC3H1 myogenic cells

Mouse BC3H1 myogenic cells and a bi-functional chemical cross linking reagent were utilized to investigate the polymerization of newly-synthesized vascular smooth muscle (alpha-actin) and non-muscle (beta- and gamma-actin) actin monomers into native F-actin filament structures during myogenesis. Two actin dimer species were identified by SDS-PAGE analysis of phenylenebismaleimide-cross linked fractions of BC3H1 myoblasts and myocytes. P-dimer was derived from the F-actin-enriched, detergent-insoluble cytoskeleton. Pulse-chase analysis revealed that D-dimer initially was associated with the cytoskeleton but then accumulated in the soluble fraction of lysed muscle cells that contained a non-filamentous or aggregated actin pool. Immunoblot analysis indicated that non-muscle and smooth muscle actins were capable of forming both types of dimer. However, induction of smooth muscle alpha-actin in developing myoblasts coincided with an increase in D-dimer level which may facilitate actin stress fiber assembly. Smooth muscle alpha-actin was rapidly utilized in differentiating myoblasts to assemble extraction-resistant F-actin filaments in the cytoskeleton whereas non-muscle beta- and gamma-actin filaments were more readily dissociated from the cytoskeleton by an extraction buffer containing ATP and EGTA. The data indicate that cytoarchitectural remodeling in developing BC3H1 myogenic cells is accompanied by selective actin isoform utilization that effectively segregates multiple isoactins into different sub-cellular domains and/or supramolecular entities.     

The effects of anesthesia on the biodistribution of drugs in rats: a carboplatin study

PURPOSE: Anesthetics can alter the biodistribution profile of drugs and, consequently, the regional pharmacokinetics of antineoplastic drugs at the tumor site. The effect of coadministered anesthetics on the biodistribution profile of carboplatin was studied in rats. METHODS: Female Wistar rats were used to compare the effects of ketamine/xylazine, thiopental and pentobarbital on the biodistribution of 30 mg/kg radiolabelled 195mPt-carboplatin administered intravenously, with conscious rats as the control group. Blood and urine samples were collected between 5 and 120 min. RESULTS: The percentage values of the injected dose of platinum per ml (%ID/ml) in plasma at the final time-point were respectively, 0.557%, 0.156%, 0.115% and 0.086%, in pentobarbital-, ketamine/xylazine- and thiopental-injected rats, and in conscious animals. Following the same sequence of groups, the %ID/ml values of platinum in the cumulative urine were 0.001%, 0.619%, 0.184% and 0.118%, respectively. Urine output varied from very little in the pentobarbital group, to several milliliters in the other groups. CONCLUSIONS: There was an increase of almost 100-fold in total platinum uptake in the kidneys, cerebrum and cerebellum of rats receiving pentobarbital over the uptake in the control rats, whereas the biodistribution profile of the thiopental group had the least variance. These results demonstrate the importance of anesthetic selection in animal pharmacokinetic studies, as it influences the biodistribution and pharmacokinetic profile of the drug being studied.     

Neocuproine, a selective Cu(I) chelator, and the relaxation of rat vascular smooth muscle by S-nitrosothiols

1. A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and S-nitroso-glutathione (GSNO). 2. Bolus injections (10 microl) of SNAP or GSNO (10(-7)-10(-3) M) were delivered into the lumen of perfused vessels pre-contracted with sufficient phenylephrine (1-7 microM) to develop pressures of 100-120 mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre-mixed with neocuproine (10(-4) M) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10(-5) M) and then injected with either pure SNAP or GSNO. 3. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)-dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.     

Fallibility of plasma urea and creatinine as indices of renal function in diarrhoeic calves treated with conventional or nutritional oral rehydration solutions

The prevalence and severity of gingival overgrowth in organ-transplant patients medicated with cyclosporin are greater in patients concomitantly medicated with nifedipine; however, no relationship between the gingival overgrowth and any of the nifedipine pharmacological variables has been demonstrated. The study examined the effect of five nifedipine pharmacological variables (nifedipine dosage, plasma concentration and gingival crevicular fluid concentration, M1 metabolite plasma concentration and the nifedipine: M1 ratio). The effect of the nifedipine variables on the gingival overgrowth score were examined using univariate and multivariate regression analysis. Adjustment for the effect of other risk factors was made by adding the distribution of each of the nifedipine variables in turn to a stepwise regression model containing previously identified risk factors for this condition. Despite the high levels of nifedipine sequestered in the GCF, only the plasma concentration of nifedipine was identified as a risk factor for the severity of gingival overgrowth in these patients (P = 0.01) once adjusted for other known risk factors (R2 for the model = 55%).     

Purification, properties, and N-terminal amino acid sequence of a kallikrein-like enzyme from the venom of Lachesis muta rhombeata (Bushmaster)

An analytical procedure was developed to measure bromate residues in baked goods using a sequence of clean-up procedures followed by high performance liquid chromatography (HPLC) with a post-column reaction for oxidants. Deionized water was used to extract bromate from bread samples. The extract was treated with a C-18 solid phase extraction column to remove lipids, a cation exchange column with the silver cation to remove chloride, and an ultrafiltration membrane to remove proteins. Further treatment of the extract with the sodium form of a propylsulphonic acid ion exchange column was necessary to remove the silver that leached from the silver column. The method had a detection limit of 3 ng/g in baked goods. Recoveries of bromate from breads ranged from 73 to 86% at a fortified bromate level of 5-100 ng/g. Pullman-type white bread, produced by a sponge and dough method, was prepared in our laboratory for measurement of residual bromate. The dough was scaled in three different weights at different specific volumes (3.8, 4.1, 4.3), and samples of each of the three weights were baked for six different baking times ranging from 24 to 34 min. When bromate at a level of 25 mg/kg was added to flour, no residual bromate was detected in any of the samples, regardless of weight and baking time.     

Treatment results and prognostic factors in 101 men treated for squamous carcinoma of the penis

PURPOSE: This retrospective study was performed to assess the treatment outcome and prognostic factors in 101 men with invasive squamous carcinoma of the penis treated at the Royal Marsden Hospital between 1960-1990. METHODS AND MATERIALS: The tumor was confined to the glans penis (T1) in 79 patients, 82 were node negative (N0), and two patients had distant metastases at presentation. The histology was Grade 1 (G1) in 36, Grade 2 (G2) in 18, Grade 3 (G3) in 28, and unknown in 19 patients. Node-positive disease was commoner in patients with G3 (p = 0.02) or T2/3/4 tumors (p = 0.007). Treatment for the primary tumor was external beam radiotherapy (EBRT) in 59, interstitial brachytherapy in 13, and partial or total penectomy in 29 patients. The median dose, dose/fraction, and treatment time for EBRT was 60 Gy, 2 Gy/fraction, and 46 days, respectively. Eighty patients received no inguinal node treatment, 13 had EBRT (4 with chemotherapy), and 8 underwent groin dissection at presentation. RESULTS: During a median follow-up of 5.2 years (2 months-22 years), 56 patients died (penile cancer 31, intercurrent illness 23 and unknown cause 2), giving 10 year overall and cause-specific survival (CSS) of 39 and 57%, respectively. Adverse prognostic factors for CSS on univariate analysis were G3, ulcerative/fungating or T2/3/ 4 tumors, node positive, Jackson's Stage 2/3/4, and surgical treatment for the primary. All but the last two were significant independent prognostic factors for CSS on multivariate analysis. Penile or perineal recurrence or residual disease after initial treatment was seen in 36 out of 98 evaluable patients, giving a 10-year local failure rate (LFR) of 45%. Local failure after initial treatment was successfully salvaged in the majority (26 out of 36) of patients with further surgery or radiotherapy, and local control was achieved ultimately in 74 out of 77 T1, 7 out of 12 T2; 3 out of 3 T3, and 3 out of 5 T4 tumors. In the 44 evaluable patients with T1 tumors treated by EBRT the only adverse RT parameter approaching prognostic significance (p = 0.052) was a BED value corrected for recovery of <60 Gy (alpha/beta 10, K = 0.5 Gy/day, mean = 21 days). CONCLUSION: Invasive squamous carcinomas of the penis carry a significant risk of loco-regional recurrence after initial radiotherapy and this can be successfully salvaged in most patients with further treatment. This mandates close follow-up to detect loco regional recurrence early.     

Diagnostic accuracy of a rural live video telepathology system

Accuracy of diagnoses rendered using a live video telepathology network was assessed for permanent sections of surgical pathology specimens. To determine accuracy, telepathology diagnoses were compared with those obtained by directly viewing the glass slide using a standard microscope. A total of 294 cases were read via both telepathology and glass slide by attending pathologists at a tertiary care medical center. Overall accuracy was defined as exact concordance between diagnoses. Clinically insignificant differences in diagnoses were excluded to determine clinically significant accuracy. For the 285 cases with complete data, the overall accuracy for telepathology was 0.912 (95% confidence interval [CI], 0.872-0.941), whereas the overall accuracy for glass slide readings was 0.968 (95% CI, 0.939-0.985). This difference is statistically significant (p = 0.009). When focusing on clinically significant discrepancies, where the difference in diagnosis might affect therapeutic decisions, the video accuracy was only slightly less than the glass slide accuracy (0.965 [95% CI, 0.934-0.982] vs. 0.982 [95% CI, 0.957-0.994], respectively), but this difference is not statistically significant (p = 0.302). Most of the cases with clinically significant differences involved lesions with inherently high interobserver variation. Certainty of diagnosis did not differ between video and glass slide readings (p = 0.911), but there was an association between certainty of diagnosis and diagnostic accuracy for video (p = 0.003 for clinically significant accuracies). Based on these findings, we recommend when using this telepathology system that only preliminary diagnoses should be given in the following situations: for diagnostic areas with known high interobserver variability; when the consultant has any degree of uncertainty about the presence or absence of the lesion in question; and when there is insufficient experience using telepathology as a diagnostic medium.