Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl−/HCO 3 − double exchange and Na+−Cl− symport

Summary Cl^– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to³⁶Cl^– and³H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN^– which immediately and completely inhibits Cl^– entry into the cell. Cellular influx was equal to 16.7eq cm^–2 hr^–1 and decreased to 8.5eq cm^–2 hr^–1 upon removal of HCO ₃ ^– from the bathing media and by bubbling 100% O₂ for 45 min. When HCO ₃ ^– was present, cellular influx was again about halved by the action of 10^–4 m acetazolamide, 10^–5 to 10^–4 m furosemide, 10^–5 to 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10^–3 m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO ₃ ^– was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO ₃ ^– was present in the salines, Na⁺ removal from the mucosal side caused a slow decline of cellular Cl^– influx; conversely, it immediately abolished cellular Cl^– influx in the absence of HCO ₃ ^– . In conclusion, about 50% of cellular influx is sensitive to HCO ₃ ^– , inhibitable by SCN^–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na⁺. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN^–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na⁺. Thus, about 50% of apical membrane NaCl influx appears to result from a Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchange, whereas the residual influx seems to be due to Na⁺–Cl^– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.     

Matric potential evaluations and measurements for gelled substrates

A new method for evaluating the matric potential of gelled media has been developed. The method allows the derivation of the matric potential as a limit of a series of measurements of water potential values from gelled media prepared without added components, from agar powders progressively cleaned of mineral impurities. Three commercial agar brands were tested, and for these the matric potential was found to contribute only between 1 and 2% of their total water potential. Thermodynamic features relating matric and osmotic potentials are described. New hypotheses for understanding the water flux mechanism from gel to tissue cultured explants are discussed. Movement of water along polymeric chains is postulated to be a facilitated step in comparison with bulk movement.     

Harmonization of the Practice of Independent Ethics Committees in Italy: Project E-Submission

Aim

The high variability of “centre-specific” documentation required by Independent Ethics Committee (IEC) plays a role in the time required for activation of participating centres of multicentre clinical trials. This study (a) provides a picture of the different activities, structural requirements and resources dedicated to the operation of the local IEC in Italy; (b) defines a detailed list of “centre-specific” documents considered as essential by the IEC for issuing its opinion and (c) suggests a “single document” to reduce the variability of the “centre-specific” documents required by the IEC.

Methodology

Two surveys were conducted through the portal of National Monitoring Centre of Clinical Trials (https://oss-sper-clin.agenziafarmaco.it/). The first survey focused on the local IEC resources and on the “centre-specific” documentation that local IEC required from the Sponsor and local Principal Investigator (PI). The second focused on “single document” required in the form of statements from the Sponsor and the PI. Answers were discussed and extended during regular scheduled teleconferences and plenary meeting.

Principal Findings

From 22/07/2009 to 15/12/2009, and from 19/04/2010 to 14/05/2010, 131 and 125 IECs responded to the first and the second surveys, respectively. 67% and 51% of IECs consider the structural requirements and the staff dedicated to the activity of the IECs as sufficient, respectively. Most of the IECs consider the “centre-specific” documentation as necessary for issuing the opinion, and a high percentage of IECs consider the proposed documentation as acceptable in substitution to any other “centre-specific” documentation already in use.

Conclusions

The harmonization of IECs practice in Italy is the first step to facilitate multicentre clinical trials. Similar efforts should be directed to reduce the total number of IECs and to standardize clinical trials approval procedures, focusing on administrative procedures as well.     

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum “XerumFree? XF205” (XF); (3) CnT-20 medium supplemented with “XerumFree? XF205” (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ?Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ?Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ?Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.     

Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.