A new system has been developed for measuring the forces produced by a small number (less than 5-150) of myosin molecules interacting with a single actin filament in vitro. The technique can resolve forces of less than a piconewton and has a time resolution in the submillisecond range. It can thus detect fluctuations of force caused by individual molecular interactions. From analysis of these force fluctuations, the coupling between the enzymatic ATPase activity of actomyosin and the resulting mechanical impulses can be elucidated.

Apolipoprotein AIV (apo AIV) is a satiety protein secreted by the small intestine. We demonstrate for the first time that apo AIV protein and apo AIV mRNA are present in rat hypothalamus, a site intimately involved in the integration of signals for regulation of food intake and energy metabolism. We further characterized the regulation of hypothalamic apo AIV mRNA levels. Food-deprived animals showed a pronounced decrease in gene expression of apo AIV in the hypothalamus, with a concomitant decrease in the jejunum. Refeeding fasted rats with standard laboratory chow for 4 h evokes a significant increase of apo AIV mRNA in jejunum but not in hypothalamus. However, lipid refeeding to the fasted animals restored apo AIV mRNA levels both in hypothalamus and jejunum. Intracerebroventricular administration of apo AIV antiserum not only stimulated feeding, but also decreased apo AIV mRNA level in the hypothalamus. These data further confirm the central role of apo AIV in the regulation of food intake.

These results indicate that strong SMF induce mutations through elevated production of intracellular superoxide radicals in E. coli.

Pradimicin A (PRM-A) is an actinomycete-derived antibiotic with the lectin-like property of being able to recognize D-mannopyranoside (Man) in the presence of Ca(2+) ion. PRM-A and its derivatives have been attracting a great deal of attention as the only family of natural carbohydrate receptors with nonpeptidic skeleton and, more recently, as conceptually novel drug candidates for human immunodeficiency virus (HIV). Despite its scientific interest and potential therapeutic importance, understanding how PRM-A recognizes Man has been severely limited. Conventional interaction analysis of PRM-A with Man in solution has been frustrated by aggregation of PRM-A and the three-component equilibrium consisting of the [PRM-A(2)/Ca(2+)], [PRM-A(2)/Ca(2+)/Man(2)], [PRM-A(2)/Ca(2+)/Man(4)] complexes, and their mixed oligomers. In this Article, we demonstrate the interaction analysis of PRM-A with methyl α-D-mannopyranoside (Man-OMe) in the solid state, which benefits from aggregate-forming propensity of PRM-A and eliminates the problem associated with the complicated equilibrium in solution. Isothermal titration calorimetry (ITC) analysis and coprecipitation experiments revealed that the primary Man binding of PRM-A is markedly tighter than the secondary one, leading to preparation of the solid aggregate solely composed of the [PRM-A(2)/Ca(2+)/Man-OMe(2)] complex. The simple 1:1 complexes of biosynthetically (13)C-enriched PRM-As and [(13)C(6)]Man-OMe facilitated the analysis of the primary Man binding of PRM-A by two-dimensional dipolar-assisted rotational resonance (2D-DARR), which clearly identified that the cavity consisted of D-alanine moiety and ABC rings of PRM-A is the Man binding site. Interestingly, the proposed Man binding site of PRM-A seems to resemble the typical architecture of artificial carbohydrate receptors.

The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high temperatures. We have previously found that replacement of the entire ␣-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic temperatures, both in vivo and in vitro, whereas the former and latter half-replacements of the ␣-helix 6 (SP4 and SP5 mutants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of ␣-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the ␣-helix 6 and loop 6 regions was important to increase the catalytic activity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5-V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures.The Calvin-Benson-Bassham (CBB) 4 cycle contributes to the fixation of carbon for all plants, algae, and cyanobacteria. The most important enzyme in the CBB cycle is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) (1, 2). Rubisco is responsible for the single carbon dioxidefixing step of the cycle, in which ribulose-1,5-bisphosphate reacts with CO 2 and H 2 O to form two molecules of 3-phosphoglycerate. However, it also catalyzes a competitive oxygenase reaction, by which ribulose-1,5-bisphosphate reacts with O 2 to form one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycolate (3, 4). Moreover, a typical Rubisco is able to fix only three carbon dioxide molecules/s, an extremely low turnover rate for an enzyme (3). Rubisco is thus considered to be the rate-limiting enzyme of the CBB pathway, and has been an important target for protein engineering (3).From sequence alignment, Rubiscos can be classified into four groups, types I, II, III, and IV (5). The classical type I and II Rubiscos function in the CBB cycle. Type I Rubisco is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L 8 S 8 ) (6). Type II Rubisco is usually composed of only two L subunits (L 2 ) (7, 8). In both cases, the L 2 dimer is the catalytic unit of Rubisco, generating two catalytic...