An NMR study of the conformation and thermodynamics of the circular dumbbell d [formula: see text] Slow exchange between two- and four-membered hairpin loops.

The circular DNA decamer 5'-d [formula: see text] 3' is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5'-GTTC-3' loop is disrupted, whereas the opposite 5'-CTTG-3' loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.     

Re-plumbing in a Mediterranean sponge

Observations are reported for Dysidea avara sponges where once functioning oscula (outlets) are converted through internal re-plumbing into functioning oversized ostia (OSO; inlets). Flow tank studies employed high-speed photography and particle tracking of laser-illuminated 0.5-6.0 microm diameter glass beads to trace particles streaming into OSO. A fluorescein dye/glass bead uptake experiment showed that an oversized ostium was connected through internal structures to the lone osculum. Beginning 30 s after uptake and continuing over a 20 min period, dye streamed from the osculum, but no beads emerged. Scanning electron microscopy revealed that beads were deposited only on the inhalant side of particle filtering choanocyte chambers and not on the exhalant side, suggesting that internal re-plumbing had occurred. Functioning OSO were also found on freshly collected specimens in the field, making it highly unlikely that formation of OSO was only an artefact of sponges being held in a laboratory tank.     

Physiological compartmentation in gonadal tissue of the common carp (Cyprinus carpio L.)

Summary Physiological compartmentation in carp (Cyprinus carpio L.) gonads was investigated after intracardial injection of horseradish peroxidase (HRP) and two mouse anti-carp-sperm monoclonal antibodies.Immunohistochemistry revealed that a physiological barrier exists in carp testis for HRP and mouse IgG monoclonal antibody around the central lumina of the tubules in which the spermatozoa are located, but not around the cysts containing the precursor germ cells. The results with HRP were confirmed by electron microscopy. Mouse IgM monoclonal antibody did not penetrate the spermatogenic cysts. Probably because of its large size, it was almost exclusively located inside blood capillaries and only sparsely in the interstitial tissue.In the ovary, HRP was regularly distributed in the gonadal tissue, whereas the IgG antibody was predominantly localised on oogonia and early prophase oocytes. The results indicate that in contrast with the testis, no barrier around germ cells exists in the carp ovary.     

A study on cell lineage,especially the germ cell line,in embryos of the teleost fish,Barbus conchonius

Summary Lucifer Yellow-Dextran labelling of lower layer cells (LLC), sometimes together with upper layer cells (ULC), of the 64-cellBarbus conchonius embryo resulted in labelled primordial germ cells (PGCs) at 12 h after fertilization (a.f.) in about 25% of cases. The presence of labelled PGCs was independent of the location of the injected blastomere with respect to the later orientation of the embryonic axis. After injection of an ULC alone, however, labelled PGCs were never found. Also, the distribution of labelled somatic cells differed between the ULC- and LLC-injected embryos. When we found fluorescent PGCs, only a few of them were labelled, suggesting that either a single predecessor exists earlier than the 64-cell stage or that the formation of germ cells is a polyclonal process. Tracing the fluorescent cells at successive stages of development shows an extensive mixing with unlabelled cells during the epiboly stage, which might well be the cause of partly unpredictable cell lineages. The chance of being committed to a specific fate is different for the ULC and LLC descendants. This might be due to relatively limited cell mixing between these two cell populations.     

Endurance exercise differentially stimulates heart and axial muscle development in zebrafish (Danio rerio)

Mechanical load is an important factor in the differentiation of cells and tissues. To investigate the effects of increased mechanical load on development of muscle and bone, zebrafish were subjected to endurance swim training for 6 h/day for 10 wk starting at 14 days after fertilization. During the first 3 wk of training, trained fish showed transiently increased growth compared with untrained (control) fish. Increased expression of proliferating cell nuclear antigen suggests that this growth is realized in part through increased cell proliferation. Red and white axial muscle fiber diameter was not affected. Total cross-sectional area of red fibers, however, was increased. An improvement in aerobic muscle performance was supported by an increase in myoglobin expression. At the end of 10 wk of training, heart and axial muscle showed increased expression of the muscle growth factor myogenin and proliferating cell nuclear antigen, but there were major differences between cardiac and axial muscle. In axial muscle, expression of the "slow" types of myosin and troponin C was increased, together with expression of erythropoietin and myoglobin, which enhance oxygen transport, indicating a shift toward a slow aerobic phenotype. In contrast, the heart muscle shifts to a faster phenotype but does not become more aerobic. This suggests that endurance training differentially affects heart and axial muscle.     

Non-radioactive in situ hybridization for the detection of cytomegalovirus infections

Acetylaminofluorene (AAF) modified cytomegalovirus (CMV) DNA probes have been applied for the rapid detection of CMV genomes by non-radioactive in situ hybridization in routinely obtained pathological material. To establish proper protocols, AAF modified mouse satellite DNA and mouse liver were used to investigate the procedural variables. Among these were type and time of fixation, glass slide coating for improved tissue adherence, protease permeabilization of sections, type and time of denaturation and hybridization, probe concentration, post-hybridization washing conditions and immunocytochemical detection. This research has led to a user-friendly procedure which, in addition to cells displaying a cytopathological effect typical for CMV infection, detects with high sensitivity CMV carrying cells that show no histo-pathological alterations. It can be readily applied in routine clinical-diagnostic laboratories.     

Thermodynamics of melting of the circular dumbbell d〈pCGC-TT-GCG-TT〉

The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson–Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5′-d〈pCGC-TT-GCG-TT〉-3′( I ) and reference compounds 5′-d〈pGGC-TT-GCC-TT≤-3′( II ) and 5′-d(GCG-TC-CGC)-3′( III ) are studied by means of nmr spectroscopy. Molecules I and II adopt dumbbell structures closed on both sides by a two-membered hairpin hop. At low temperature I consists of a mixture of two slowly exchanging forms, denoted L2L2 and L2L4 . The low-temperature L2L2 form is the fully intact minihairpin structure with three Watson–Crick C-G base pairs. The high-temperature form, L2L4 ,contains a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop, with the cytosine base placed in a syn orientation. The opposite 5′-CTTG-3′ loop remains stable. A study of the noncircular hairpin structure III shows similar conformational behavior for the 5′-GTTC-3′ loop as found in I a syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point T_m of II was estimated to lie above 365 K. The T_m value of the duplex stem and the 5′-CTTG-3′ loop of the L2L4 form ofIis 352 ± 2 K. The ΔH° is calculated as ?89 ± 10 kJ/mol. The T_m value determined for the individual residues of the 5′-GTTC-3′ loop lies 4°–11° lower. The enthalpy ΔH° of melting the thymine residues in the 5′-GTTC-3′ loop is calculated to be -61± 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for ΔH° of +30 kJ/mol in going from a two-memberedto a four-membered loop, in agreement with the enthalpy difference of +28 k.j/mol between the two loops at the T_m midpoint. For hairpin III the upper limit for ΔH° going from a two-membered to a four-membered loop amounts to ±21 kJ/mol. The mutual exchange rate between the L2 and L4 form in III is estimated as 13.6 s^?1. Our results clearly suggest that small four-way DNA junctions(model for immobilized Holliday junctions) can be designed that consist of a single DNA strandthat features -CTTG-caps on three of the four arms of the junction. © 1995 John Wiley & Sons, Inc.     

Characterization of two novel human small heat shock proteins: protein kinase-related HspB8 and testis-specific HspB9

Some binary mixtures of cholesterol and phospholipids in monolayers have thermodynamic phase diagrams with two upper miscibility critical points. This feature has been interpreted in terms of 'condensed complexes' between the phospholipid and cholesterol. The present work gives evidence for the formation of complexes with a common simple integral stoichiometry in binary mixtures of cholesterol and a series of five sphingomyelins where the amide-linked acyl chain length is varied. This indicates that these complexes have a distinct geometry even though they form a liquid phase.     

Solid-phase synthesis of an RNA nucleopeptide fragment from the nucleoprotein of poliovirus.

The naturally occurring RNA-nucleopeptide H-Ala-Tyr[5'-pUUAAAAC-3']-NH2 is prepared via a solid-phase phosphite triester approach using N-SiOMB/O-TBDMS-protected nucleosides. Preliminary 1H-NMR studies show that the peptidyl unit has a remarkable effect on the conformational behaviour of the RNA moiety in the nucleopeptide.