A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.     

Evaluation of the efficacy of various hypotensive drugs in broad-breasted white turkeys as an experimental model of arterial hypertension with high catecholamine levels

Male Broad Breasted White Turkeys (BBWT) represent an experimental model of arterial hypertension characterized by high levels of circulating and tissue catecholamines. We thought interesting to evaluate the efficacy of the long term treatment with different antihypertensive drugs. 59 male BBWT were studied, divided in five groups. The first group (13 animals) was treated with placebo; the second (14 animals) with oxprenolol 4 mg/Kg/q.d.; the third (10 animals) with labetalol 25 mg/Kg/q.d.; the fourth (11 animals) with verapamil 15 mg/Kg/ q.d.; the fifth (11 animals) with captopril 8 mg/Kg/ q.d. and furosemide 2,5 mg/Kg/q.d. All drugs were given p.o, from the 8th to the 33rd week of age. Weekly or every two weeks Blood Pressure (BP) and Heart Rate (HR) were measured by an indirect method. In all animals BP progressively increased and HR progressively decreased with age. Only the labetalol-treated animals showed a significant reduction of BP and HR through the study period as compared with the placebo-treated animals. These results confirm the preminent role played by the high levels of circulating catecholamines in determining and maintaining the arterial hypertension.     

Diversity and distribution of reptiles in Romania

The reptile fauna of Romania comprises 23 species, out of which 12 species reach here the limit of their geographic range. We compiled and updated a national database of the reptile species occurrences from a variety of sources including our own field surveys, personal communication from specialists, museum collections and the scientific literature. The occurrence records were georeferenced and stored in a geodatabase for additional analysis of their spatial patterns. The spatial analysis revealed a biased sampling effort concentrated in various protected areas, and deficient in the vast agricultural areas of the southern part of Romania. The patterns of species richness showed a higher number of species in the warmer and drier regions, and a relatively low number of species in the rest of the country. Our database provides a starting point for further analyses, and represents a reliable tool for drafting conservation plans.     

Identification of a developmentally regulated calcium-binding protein in Trypanosoma brucei

Calcium ions mediate cellular activity by binding to specific cellular proteins. The following study systematically examines the cellular complement of calcium-binding proteins in different cell fractions and life cycle stages of Trypanosoma brucei. Using a 45Ca-gel overlay procedure, eight calcium-binding proteins were consistently observed. The majority of proteins were cytosolic (84, 70, 64, 22, and 15 kd) while the remainder (55, 46, and 29 kd) were particulate. Although calmodulin was detected amongst the calcium-binding proteins, it did not represent the majority of calcium-binding activity. Of special interest was the 46 kd calcium-binding protein which was associated with 3-fold more calcium in cultured procyclic forms than in slender bloodstream forms. By contrast, promastigote forms of Leishmania mexicana did not contain the 46 kd calcium-binding protein. These data suggest that responsiveness to calcium signals may vary during the trypanosome life cycle as a result of changes in the cellular complement of calcium-binding proteins.     

Structural determinants of cationic amphiphilic amines which induce clear cytoplasmic vacuoles in cultured cells

Disobutamide (D), an antiarrhythmic cationic amphiphilic amine (CAA), was withdrawn from clinical testing when clear cytoplasmic vacuoles (CCV) were found in the rat and dog during toxicity studies. To delineate the structural determinants of amines that induce CCV, we exposed cultured rat urinary bladder carcinoma and rabbit aorta muscle cells to numerous cationic drugs and chemicals and examined cells by phase light microscopy. The cationic moiety of these CAA was responsible for the induction of CCV. The very potent inducers were compounds that had two strongly basic amine (cationic) centers. The bis tertiary amines were particularly potent inducers. Aliphatic diamines of minimal lipophilicity-induced CCV, thus showing that an "amphiphilic" structural feature, though present in many CAA drugs, is not necessary for CCV induction. The distance between the two cationic centers was irrelevant to the induction of CCV. These results support the concept that CCV are a manifestation of intracellular (e.g., intralysosomal) drug storage. These structural delineations will be useful in future drug design and for further understanding of drug-cell interactions. Based on these findings, we were able to synthesize an antiarrhythmic CAA which did not induce CCV.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

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.     

Expression of HOX homeogenes in human neuroblastoma cell culture lines

Mammalian genes containing a class-I homeobox (HOX genes) are highly expressed in the embryonic nervous system. As a first step towards the molecular analysis of the role these genes play in neural cells, we studied the expression of four human HOX genes in five neuroblastoma (NB) cell lines - SK-N-BE, CHP-134, IMR-32, SK-N-SH and LAN-1 - during the process of differentiation induced by treatment with retinoic acid (RA). The four genes, HOX1D, 2F, 3E and 4B, located at corresponding positions in the four HOX loci, share a high degree of sequence similarity with the Drosophila Deformed homeotic gene and constitute a homology group, group 10. One of these genes, HOX1D, is not expressed in the cells used, whereas the other three are highly expressed in untreated and RA-induced NB cells, even though the expression pattern in the various lines is slightly different for the three genes. Our analysis reveals a complex and specific expression pattern in these lines, paving the way to an identification of different NB-cell populations by means of specific HOX gene expression schemes. On the other hand, in every line studied, morphological maturation toward a neuronal differentiated phenotype appears to be associated with increased HOX gene expression.