Imaging biomolecule arrays by atomic force microscopy.

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Characterization of plant satellite DNA using restriction nucleases

The structural organization of satellite DNAs of mustard Brassica nigra and lemon Citrus limon has been studied by digestion with restriction nucleases. Analysis of DNA products produced by EcoRI and Bam I shows that two satellite DNAs contain long range periodicities belonging to several repeated sequences. The periodicities in two satellite DNAs differ characteristically, however, they have been found to contain common homologous sequences. Using the restriction nuclease Bsp I, a highly periodical fractions has been found in Citrus satellite DNA, composed of Bsp I fragments ranging from 80 to 1240 basepain. The major repeat units comprise five Bsp I fragments ranging from 80 to 200 bp. These fractions characterized by a high content of 5-methyl-cytosine.     

Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.     

Enzymic in vitro repair and chemical nature of DNA chain breaks induced by incorporated phosphorus-32P decay.

In vitro repair of single strand breaks in T4 and phage DNA caused by 32p decay was studied. Zone centrifugation procedure showed that polynucleotide kinase, ligase enzyme system failed to repair 32P-damages. It was found that damaged DNA contained gaps and could be repaired by DNA-polymerase I, polynucleotide ligase treatment.     

Relationship of drinking water quality (hardness-softness) to cardiovascular mortality in Newfoundland

The profile of mortality in Newfoundland was analysed for all deaths occurring in 1969 of persons 35 to 69 years of age, of whom the total was 1036. An exceptionally high cardiovascular mortality (793 deaths/100,000) was noted for St. John''s, the capital city of Newfoundland, a city which has an extremely soft drinking-water supply. This high rate corresponds to that observed in the “high mortality belt” reported for the east coast of the United States, and in conjunction with data from mainland Canada, extends the belt across the entire eastern aspect of North America. The proportion of cardiovascular deaths of men occurring outside the hospital was less within hard drinking-water areas in Newfoundland than in the soft water areas of the province. Thus, the statistics reported here of cardiovascular mortality confirm evidence reported elsewhere on “macro-geographic” variations in this disease(s) as well as “micro-geographic” regional variations which may be dependent upon local environmental factors.     

Effects of alcohol mixed with energy drink and alcohol alone on subjective intoxication

Recent studies suggest that the combination of caffeine-containing drinks together with alcohol might reduce the subjective feelings of alcohol intoxication—the so-called “masking effect”. In this study, we aimed to review the effects of alcohol in combination with caffeine or energy drink with special focus on the “masking effect”. Fifty-two healthy male volunteers were analysed concerning breath alcohol concentration and subjective sensations of intoxication using a 18 item Visual Analogue Scale in a randomised, double-blinded, controlled, four treatments cross-over trial after consumption of (A) placebo, (B) alcohol (vodka 37.5 % at a dose of 46.5 g ethanol), (C) alcohol in combination with caffeine at a dose of 80 mg (equivalent to one 250 ml can of energy drink) and (D) alcohol in combination with energy drink at a dose of 250 ml (one can). Primary variables were headache, weakness, salivation and motor coordination. Out of four primary variables, weakness and motor coordination showed a statistically significant difference between alcohol and non-alcohol group, out of 14 secondary variables, five more variables (dizziness, alterations in sight, alterations in walking, agitation and alterations in speech) also showed significant differences due mainly to contrasts with the non-alcohol group. In none of these end points, could a statistically significant effect be found for the additional ingestion of energy drink or caffeine on the subjective feelings of alcohol intoxication. This within-subjects study does not confirm the presence of a “masking effect” when combining caffeine or energy drink with alcohol.