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.     

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.     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage.

A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.     

Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.