Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Oxygen metabolism,oxidative stress and acid-base physiology of dental plaque biofilms

Dental plaque is a natural biofilm which has been a focus of attention for many years because of its known roles in caries and periodontal diseases. Acid production by plaque bacteria leads to the erosion of tooth mineral in caries, and the cariogenicity of plaque is related to population levels of acid-tolerant organisms such as mutants streptococci. However, the biofilm character of plaque allows for survival of a diverse flora, including less acid-tolerant organisms, some of which can produce ammonia from arginine or urea to counter acidification. Plaque is often considered to be relatively anaerobic. However, evidence is presented here that both supragingival and subgingival plaque have active oxygen metabolism and that plaque bacteria, including anaerobes, have developed defenses against oxidative stress. Even in subgingival plaque associated with periodontitis, measured residual oxygen levels are sufficient to allow for oxygen metabolism by organisms considered to be extremely anaerobic such asTreponema denticola, which metabolizes oxygen by means of NADH oxidases and produces the protective enzymes superoxide dismutase and NADH peroxidase. The finding that plaque bacteria produce a variety of protective enzymes is a good indicator that oxidative stress is a part of their everyday life. The biofilm character of plaque allows for population diversity and coexistence of aerobes, anaerobes and microaerophiles. Overall, agents that affect oxidative metabolism offer possibilities for reducing the pathogenic activities of plaque.     

Chromosomal localization of a tandemly repeated DNA sequence in Trifolium repens L

INTRoDUCTIoNlYho1iumrePensL,whiteclover,isaneconomicallyimportantplantspeciesintemperatepastures.Asbrieflyreportedby[1],ithas16pairsofchromosomes(2n=32).Asyet,nodetailedcytologicalexaminationofthisspecies,suchasC-banding,hasbeenrep0rted.Inthelastdecade,thetechnique0fC-bandinghasbeenusedt0examinehighlyrepeatedsequencesinplantchrom0s0mesandhasprovidedausefultoolf0rtheanalysis0fcyt0geneticstructureincr0pplants[2-71.Inplants,thechr0m0s0mall0calizationofhighlyrepeatedDNAsequencesbyinsituhybr…     

Antityphoid and antitetanic intradermal vaccination of chronic neuropsychic patients in a typhoid fever outbreak.

Intradermal immunization of 229 chronic neuropsychic patients in Gura Ocnitzei Sanatorium, D?mbovitza County, where a typhoid fever outbreak burst, was performed with a freeze-dried typhoid vaccine, suspended in purified and concentrated tetanic anatoxin. Adverse reactions were minimal and immunologic response, evaluated by laboratory tests, was good, reaching the level of that obtained as a result of classic vaccination methods (subcutaneous antityphoid and intramuscular anti-tetanic). Both after vaccination and also booster O and H agglutinating titers increased 4-7 times and anti-S. typhi. seroprotecting effect reached and maintained a high level. Moreover, antityphoid vaccination permitted epidemic focus limiting and its complete extinguishing after booster. Antitetanic vaccination resulted in protecting titers settlement (greater than or equal to 0.01 IU/ml) in all previously non-protected persons (1/3 of all subjects) and in the increase of protecting titers, existing before vaccination in the other persons.