Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Solid-state modifications of poly(γ-ethyl-L-glutamate)

Several modification of the arrangements of α-helical molecules were found in the solid films of poly (γ-ethyl-L -glutamate), depending on the casting solvent and the temperature. The helical conformation is somewhat looser than the normal 18-residue, 5-turn α-helix. Using x-ray diffraction, the types of molecular arrangements were classified into tetragonal, pseudohexagonal, and hexagonal ones. Tetragonal packing was observed in the filmm (form T) prepared by casting the solution in trifluorethanol or dichlorethane. The sample obtained from chloroform solution is a well-ordered, pseudohexagonal modification (form I). Forms I and T change into a poorly crystalline form III by annealing at temperatures above 130° C. It is particularly noteworthy that the less-ordered form III exhibits a thermoreversible transition around 110°C into a well-ordered form H with the hexagonal molecular packing.     

Seasonal changes in inhibin activity in seminal plasma and serum concentrations of FSH, LH and testosterone in the male goat (Capra hircus)

Inhibin activity in goat seminal plasma was measured by in vitro assay throughout successive 9 months and its relationship with the serum FSH, LH and testosterone concentrations was investigated. Total inhibin activity (TIA) in seminal plasma gradually increased from spring to summer, reduced in autumn (P<0.05) and recovered toward winter (P<0.05). Serum FSH and LH reached a peak in mid-summer (P<0.01) and returned to the low levels in autumn. Serum testosterone also increased in mid-summer and kept the high levels until the early winter (P<0.05). Some positive correlation was found in monthly levels between seminal TIA and serum FSH (r=0.305; P<0.05). Results suggest that the summer increase of inhibin activity in seminal plasma relates with the mid-summer rise of serum FSH levels in the male goat.     

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Calcium influx in a single rat basophilic leukemia cell as revealed with a digital imaging fluorescence microscope

Using a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37 degrees C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

Depolarization induced by injection of cyclic nucleotides into frog taste cell

In order to identify the intracellular transmitter involved in the taste transduction process, cyclic nucleotides were iontophoretically injected into the frog taste cells while membrane potentials were recorded intracellularly. Injection of either cyclic GMP or cyclic AMP induced a depolarization response of about 5 mV in the taste cells, but injection of Cl- had no effect. The rate of a repolarization after the depolarization elicited by cyclic GMP was larger than that after cyclic AMP. The possible role of cyclic nucleotide in the taste transduction was discussed.     

Centrifugal decrement in diameter of myelinated afferent fibres innervating frog taste organ

1. Regional changes in the diameter of single myelinated afferent nerve fibres innervating the taste disc of the fungiform papillae on the bullfrog tongue were investigated morphologically and functionally. 2. The diameter of myelinated afferents in the medial lingual branch of the glossopharyngeal nerve averaged 8.4 microns at the proximal end of the tongue and gradually decreased at the rate of 0.8 micron/cm length of the fibres as they ran in the apical direction of the tongue. 3. The conduction velocity of single myelinated afferent fibres within the tongue decreased gradually as they ran peripherally. 4. Electrophysiological inspection of neural connections between the fungiform papillae suggests that a gradual centrifugal decrease in the diameter of a single myelinated afferent fibre is not due to multiple bifurcations of the fibre at various sites within the tongue, but due to a natural gradual decrease in the thickness of the myelin sheath and the diameter of axon.