Molecular dynamics of alamethicin transmembrane channels from open-channel current noise analysis.

Conductance noise measurement of the open states of alamethicin transmembrane channels reveals excess noise attributable to cooperative low-frequency molecular dynamics that can generate fluctuations approximately 1 A rms in the effective channel pore radius. Single-channel currents through both persistent and nonpersistent channels with multiple conductance states formed by purified polypeptide alamethicin in artificial phospholipid bilayers isolated onto micropipettes with gigaohm seals were recorded using a voltage-clamp technique with low background noise (rms noise < 3 pA up to 20 kHz). Current noise power spectra between 100 Hz and 20 kHz of each open channel state showed little frequency dependence. Noise from undetected conductance state transitions was insignificant. Johnson and shot noises were evaluated. Current noise caused by electrolyte concentration fluctuation via diffusion was isolated by its dependence on buffer concentration. After removing these contributions, significant current noise remains in all persistent channel states and increases in higher conductance states. In nonpersistent channels, remaining noise occurs primarily in the lowest two states. These fluctuations of channel conductance are attributed to thermal oscillations of the channel molecular conformation and are modeled as a Langevin translational oscillation of alamethicin molecules moving radially from the channel pore, damped mostly by lipid bilayer viscosity.     

Structure and function of adenovirus type 12 defective virions.

Purified human adenovirus type 12 preparations contain defective virions with a lighter density. These defective virions were isolated, and their biological functions and DNA were characterized. They can induce early and late antigens in infected cells and tumors in newborn hamsters with similar efficiency as complete virions. The majority of the DNA molecules from light virions contain deletions mapping near 16% from the left-hand end of the genome. Mechanisms for the generation of these molecules are discussed.     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.     

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.     

Genetics of glucosephosphate isomerase in Aedes togoi (Diptera: Culicidae)

The genetics of glucosephosphate isomerase (E.C. 5.3.1.9) in two strains (Malaysian and Taiwan) of Aedes togoi is reported. Three electrophoretic phenotypes were present in both sexes. The zymogram patterns were identical in both strains of A. togoi. The phenotypes were governed by a pair of codominant alleles. The allele frequency of the slow-moving band was 0.63 in the Malaysian strain and was 0.86 and 0.82 in F161 and F169 generations, respectively, of the Taiwan strain. The sample studied was in good accord with Hardy-Weinberg expectations.     

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.