Fine needle aspiration cytology of hepatoblastoma. Recognition of subtypes on cytomorphology

OBJECTIVE: To delineate the cytomorphologic appearances of hepatoblastoma (HBL) in the largest series to date and to evaluate the feasibility of subtyping on fine needle aspiration cytology (FNAC). STUDY DESIGN: Papanicolaou- and May-Grünwald-Giemsa-stained smears of aspirates from 26 cases of HBL were analyzed by 2 observers. Histologic material, available in 15 cases, was correlated. A cytology grouping system was proposed on the basis of which all cases were classified. RESULTS: The ages of the patients ranged from 4 months to 9 years. Twenty-five cases were categorized as epithelial HBL, with epithelial fragments showing a trabecular arrangement and acinar formation in all, and extramedullary hemopoiesis in 20 cases. It was possible to differentiate fetal and embryonal areas on FNAC. Six cases showed only fetal elements (cytology group F), characterized by cells with abundant cytoplasm and a small, rounded nucleus resembling a normal fetal hepatocyte. The chromatin was finely granular, with a single, central nucleolus. Pleomorphism and mitoses were not seen, and the nuclear/cytoplasmic ratio was < or = 1/3. Fourteen cases showed, in addition to fetal elements, an embryonal component characterized by cells with scant cytoplasm, a pleomorphic nucleus, N/C ratio of > or = 3/1, coarsely granular chromatin and 2-4 angulated nucleoli. Mitoses were seen in these cells (1-4/1,000 cells). Of these 14 cases, 6 showed predominantly fetal and scant embryonal cells, while 8 cases showed fetal and embryonal components in equal amounts (cytology groups Fe and FE, respectively). Four cases showed predominantly embryonal cells (cytology group E). One case was unclassifiable (U). On histology, 8 of 14 cases were of mixed epithelial and mesenchymal type, but mesenchymal tissue was not seen on the corresponding cytology. The cytology grouping system correlated well with histology. One case was small cell undifferentiated HBL and resembled a round cell tumor without differentiation. Macrotrabecular arrangement was not seen on cytology but was seen on histology in 1 case. CONCLUSION: Epithelial HBL can be easily diagnosed in aspirates further classified into fetal and embryonal subtypes, which may be of prognostic relevance. The proposed cytology grouping system is effective in semiquantification of the observed subtypes.     

A computational model of the human left-ventricular epicardial myocyte

A computational model of the human left-ventricular epicardial myocyte is presented. Models of each of the major ionic currents present in these cells are formulated and validated using experimental data obtained from studies of recombinant human ion channels and/or whole-cell recording from single myocytes isolated from human left-ventricular subepicardium. Continuous-time Markov chain models for the gating of the fast Na(+) current, transient outward current, rapid component of the delayed rectifier current, and the L-type calcium current are modified to represent human data at physiological temperature. A new model for the gating of the slow component of the delayed rectifier current is formulated and validated against experimental data. Properties of calcium handling and exchanger currents are altered to appropriately represent the dynamics of intracellular ion concentrations. The model is able to both reproduce and predict a wide range of behaviors observed experimentally including action potential morphology, ionic currents, intracellular calcium transients, frequency dependence of action-potential duration, Ca(2+)-frequency relations, and extrasystolic restitution/post-extrasystolic potentiation. The model therefore serves as a useful tool for investigating mechanisms of arrhythmia and consequences of drug-channel interactions in the human left-ventricular myocyte.     

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.     

Tumor cell alpha3beta1 integrin and vascular laminin-5 mediate pulmonary arrest and metastasis

Arrest of circulating tumor cells in distant organs is required for hematogenous metastasis, but the tumor cell surface molecules responsible have not been identified. Here, we show that the tumor cell alpha3beta1 integrin makes an important contribution to arrest in the lung and to early colony formation. These analyses indicated that pulmonary arrest does not occur merely due to size restriction, and raised the question of how the tumor cell alpha3beta1 integrin contacts its best-defined ligand, laminin (LN)-5, a basement membrane (BM) component. Further analyses revealed that LN-5 is available to the tumor cell in preexisting patches of exposed BM in the pulmonary vasculature. The early arrest of tumor cells in the pulmonary vasculature through interaction of alpha3beta1 integrin with LN-5 in exposed BM provides both a molecular and a structural basis for cell arrest during pulmonary metastasis.     

Rise of plasma ghrelin with weight loss is not sustained during weight maintenance

Objective: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects. Research Methods and Procedures: This study was a randomized clinical trial, with a 12‐month follow‐up period. Obese Mexican‐American women matched for age and BMI were randomized to a 12‐month WL program (n = 25) or no intervention (controls, n = 23). Interventions included diet, exercise, and orlistat. Body weight and fasting ghrelin, leptin, insulin, and glucose concentrations were measured at baseline and 6 and 12 months. Results: The WL group lost 8.5% of body weight after 6 months and maintained the new weight for the next 6 months. Ghrelin concentrations increased significantly at 6 months but returned to baseline at 12 months. Baseline ghrelin concentrations were directly related to the degree of WL achieved after 12 months. Controls experienced no change in BMI or ghrelin levels. There were no associations between plasma ghrelin and leptin or insulin concentrations. Discussion: Consistent with previous results, ghrelin rises in response to WL, perhaps as a counterregulatory mechanism. However, the present results indicate that ghrelin concentrations return to baseline with sustained weight maintenance, suggesting that its effects are unlikely to regulate long‐term energy balance. Baseline ghrelin concentrations are related to the degree of WL that can be achieved by active weight reduction.     

A locus for juvenile myoclonic epilepsy maps to 2q33–q36

We performed a whole genome linkage analysis in a three-generation south Indian family with multiple members affected with juvenile myoclonic epilepsy (JME). The maximum two-point LOD score obtained was 3.32 at recombination fraction (θ) = 0 for D2S2248. The highest multipoint score of 3.59 was observed for the genomic interval between D2S2322 and D2S2228 at the chromosomal region 2q33–q36. Proximal and distal boundaries of the critical genetic interval were defined by D2S116 and D2S2390, respectively. A 24-Mb haplotype was found to co-segregate with JME in the family. While any potentially causative variant in the functional candidate genes, SLC4A3, SLC23A3, SLC11A1 and KCNE4, was not detected, we propose to examine brain-expressed NRP2, MAP2, PAX3, GPR1, TNS1 and DNPEP, and other such positional candidate genes to identify the disease-causing gene for the disorder.     

Inhibition profiling of human carbonic anhydrase II by high-throughput screening of structurally diverse, biologically active compounds

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.