Sequence analysis of a 101-kilobase plasmid required for agar degradation by a Microscilla isolate.

An agar-degrading marine bacterium identified as a Microscilla species was isolated from coastal California marine sediment. This organism harbored a single 101-kb circular DNA plasmid designated pSD15. The complete nucleotide sequence of pSD15 was obtained, and sequence analysis indicated a number of genes putatively encoding a variety of enzymes involved in polysaccharide utilization. The most striking feature was the occurrence of five putative agarase genes. Loss of the plasmid, which occurred at a surprisingly high frequency, was associated with loss of agarase activity, supporting the sequence analysis results.     

Physiological responses of exercised-fatigued individuals exposed to wet-cold conditions.

Thirteen healthy and fit men [age = 27 +/- 8 (SD) yr, height = 177 +/- 5 cm, mass = 75 +/- 7 kg, body fat = 14 +/- 5%, maximal O2 consumption = 51 +/- 4 ml. kg-1. min-1] participated in an experiment designed to test their thermoregulatory response to a challenging cold exposure after 5 h of demanding mixed exercise during which only water was consumed. Subjects expended 7,314 +/- 741 kJ on cycling, rowing, and treadmill-walking machines, performed 8,403 +/- 1,401 kg. m of mechanical work during resistance exercises, and completed 120 inclined sit-ups. Subjects then assumed a seated position in a 10 degrees C air environment while wearing shorts, T-shirt, rain hat, and neoprene gloves and boots. After 30 min the subjects were showered continuously with cold water ( approximately 920 ml/min at 10 degrees C) on their backs accompanied by a 6 km/h wind for up to 4 h. Blood samples were taken from the nondominant arm every 30 min during the exposure and assayed for energy metabolites, hormones, indexes of hydration, and neurotransmitters. Counterbalanced control trials without prior exercise were also conducted. Blood insulin was higher during the control trial, whereas values of glycerol, nonesterified fatty acids, beta-hydroxybutyrate, lactate, cortisol, free triiodothyronine, and thyroxine were lower. Three subjects lasted the maximum duration of 4.5 h for control and fatigue trials, with final rectal temperatures of 36.43 +/- 0.21 and 36.08 +/- 0.49 degrees C, respectively. Overall, the duration of 172 +/- 68 (SD) min for the fatigue trial was not significantly different from that of the control trial (197 +/- 72 min) and, therefore, was not affected by the preexposure exercise. Although duration was positively correlated to body fatness and shivering intensity, the latter was not correlated to any physical characteristic or the fitness level of the individual.     

The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.     

Structure of Haze Forming Proteins in White Wines: Vitis vinifera Thaumatin-Like Proteins

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.     

Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Production of acidic xylo-oligosaccharides by a family 10 endoxylanase from Thermoascus aurantiacus and use as plant growth regulators

Neutral and acidic oligosaccharides were obtained from birchwood xylan by treatment with an endoxylanase, family 10 class, from Thermoascus aurantiacus. The main acidic xylooligosaccharide (aldotetrauronic acid) was separated from the hydrolysate by anion-exchange and size-exclusion chromatography and the primary structure was determined by ¹³C NMR spectroscopy. The aldotetrauronic yield was 15% (w/w) of the total solubilised sugars. The addition of purified aldoterauronic acid at 1.6–16 mg l^–1 growth medium, induced callus and somatic embryogenesis in culture explants of common mallow (Malva silvestris L.) and cotton (Gosssypium hirsutum).     

Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.     

Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies

The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine.