Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Overstretching of DNA occurs at about 60–70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.     

Phylogenetic analysis of 1.5 Mbp and platypus EST data refute the Marsupionta hypothesis and unequivocally support Monotremata as sister group to Marsupialia/Placentalia

The extant mammalian groups Monotremata, Marsupialia and Placentalia are, according to the 'Theria' hypothesis, traditionally classified into two subclasses. The subclass Prototheria includes the monotremes and subclass Theria marsupials and placental mammals. Based on some morphological and molecular data, an alternative proposition, the Marsupionta hypothesis, favours a sister group relationship between monotremes and marsupials to the exclusion of placental mammals. Phylogenetic analyses of single genes and even multiple gene alignments have not yet been able to conclusively resolve this basal mammalian divergence. We have examined this problem using one data set composed of expressed sequence tags (EST) and another containing 1 510 509 nucleotide (nt) sites from 1358 inferred cDNA genomic sequences. All analyses of the concatenated sequences unambiguously supported the Theria hypothesis. The Marsupionta hypothesis was rejected with high statistical confidence from both data sets. In spite of the strong support for Theria, a non-negligible number of single genes supported either of the two alternative hypotheses. The divergence between monotremes and therian mammals was estimated to have taken place 168–178 Mya, a dating compatible with the fossil record. Considering the long common evolutionary branch of therians, it is surprising that sequence data from many thousand amino acid sites were needed to conclusively resolve their relationship to monotremes. This finding draws attention to other mammalian divergences that have been taken as unequivocally settled based on much smaller alignments. EST data provide a comprehensive random sample of protein coding sequences and an economic way to produce large amounts of data for phylogenetic analysis of species for which genomic sequences are not yet available.     

Methods and Errors in Measurements of Synovial Fluid Volume in Stifles with Low Volume and High Viscosity Synovial Fluid. An Experimental Study in Goats

Synovial fluid (SF) volume was calculated using various methods in the stifles of goats, in which the cranial cruciate ligament had been transected on one side. Measurements were performed prior to surgery and again 4,8, and 18 weeks following surgery, by measuring the dilution of an injected radioactive tracer diluted by the SF. Later, 7 months following surgery, SF volume measurements using simple arthrocentesis were performed on stifles in 9 of the goats, and the SF that could not be aspirated, was calculated using 2 indirect methods simultaneously on identical fluids in 3 of these goats. SF was also collected directly during staged arthrotomy of the stifles in 4 goats. There were conflicting results between methods, but the resulting calculated SF volumes seemed to be larger in the operated stifles compared to the controls for all the methods at about the same degree. The 2 indirect methods used to calculate the fluid remaining in the joints following arthrocentesis gave disparate volume calculations. The experiments revealed sources of error in all methods. Direct methods failed to acquire the total fluid volume, and indirect methods were subject to improper mixing and escape of the injected fluid or synovial fluid or both. It was concluded that none of the methods could be used to measure the “true” volume of SF, if such a concept exists and can be defined. None of the methods were considered reliable to compare volumes in different type of joints containing this type of fluid. It was, however, concluded that all the methods gave indication of increased SF volume present on a relative basis when paired joints were compared.     

Inhibitory effects of HgCl2 on excitation-secretion coupling at the motor nerve terminal and excitation-contraction coupling in the muscle cell

Summary 1. Indirect and direct twitch (0.1-Hz) stimulation of the rat phrenic nerve-diaphragm disclosed that the inhibitory effect of HgCl₂, 3.7 × 10^–5 M, on the neuromuscular transmission and in the muscle cell, was accelerated by 10-sec periods of 50-Hz tetanic stimulation every 10 min. This activity-dependent enhancement suggested an inhibitory mechanism of HgCl₂ related to the development of fatigue, like membrane depolarization or decreased excitability, decreased availability of transmitter, or interference with the factors controlling excitation-secretion coupling of the nerve terminal, i.e. (Ca²⁺)₀ or (Ca²⁺)_i, and excitation-contraction coupling in the muscle cell, i.e., (Ca²⁺)_i.2. During both indirect and direct stimulation, HgCl₂-induced inhibition was enhanced markedly by pretreatment with caffeine, which releases Ca²⁺ from endoplasmic and sarcoplasmic reticulum in the nerve terminal and muscle cell, respectively. This caffeine-induced enhancement was completely antagonized by dantrolene, which inhibits the caffeine-induced release. However, dantrolene alone did not antagonize the HgCl₂-induced inhibition.3. Since caffeine depletes the intracellular Ca²⁺ stores of the smooth endoplasmic reticulum, HgCl₂ probably inhibits by binding to SH groups of transport proteins conveying the messenger function of (Ca²⁺)_i. In the muscle cell this leads to inhibition of contraction. In the nerve terminal, an additional enhancement of the HgCl₂-induced inhibition, by inhibiting reuptake of choline by TEA and tetanic stimulation, suggested that HgCl₂ inhibited a (Ca²⁺)_i signal necessary for this limiting factor in resynthesis of acetylcholine.4. The (Ca²⁺)₀ signal necessary for stimulus-induced release of acetylcholine was not affected by HgCl₂. Hyperpolarization in K⁺-free solution antagonized the inhibitory effect of HgCl₂ at indirect stimulation, and Ca²⁺-free solution enhanced the inhibitory effect at direct stimulation. K⁺ depolarization, membrane electric field increase with high Ca²⁺, membrane stabilization with lidocaine, and half-threshold stimulation, did not change the inhibitory effect of HgCl CH₃HgCl, 1.85 × 10^–5 M, disclosed a synergistic interaction with caffeine during direct, but not during indirect, stimulation.