Modification of frequency augmentation-potentiation by GTPγS in the frog neuromuscular junction

The effect of modifiers of guanine nucleotide-binding proteins (G proteins) on the frequency augmentation-potentiation of transmitter release were studied in the frog neuromuscular junction. Using Genetransfer^R as a carrier the mean quantal content of the endplate potential increased by penetration of GTPγS into the presynaptic nerve terminal. Neither GTPγS alone nor carrier alone had any effect. The relationship of log (mean quantal content) versus stimulation frequency changed from a single linear to a dual linear function, suggesting that the immediately releasable pool was modified. GDPβS + carrier also had similar effects, but was less potent. Aluminium fluoride was without effect. Extracellularly recorded presynaptic nerve action potentials remained unchanged with GTPγS + carrier. Also, GTPγS + carrier did not affect the action potential nor the cytosolic Ca²⁺ concentration in differentiated NG108–15 hybrid cells. It is suggested that some smg-type G protein-dependent processes are involved in determining frequency augmentation-potentiation.     

Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues

Summary Glycogen phosphorylase has at least three isoenzymes, i.e. muscle-, liver-, and brain-types. Antibodies have been raised against highly purified isoenzymes from rat muscle, liver and brain and found to react specifically to extracts from human muscle, liver and brain, respectively. Using these antibodies and the unlabelled antibody—enzyme method, each of the three isoenzymes has been localized in both rat and human tissues.     

Mechanically induced electrical and intracellular calcium responses in normal and cancerous mammary cells.

Mechanically induced channel activities and increase of intracellular calcium ([Ca2+]i) in normal and cancerous murine mammary cells (MMT 060562) were investigated using the patch clamp technique and Fura-2 fluorescence. Both cell types showed similar properties. Upon mechanical stimulation, activation of the Ca(2+)-dependent K+ channel or outward membrane current was recorded in cells which were several cells distant from the stimulated cell. Mechanical stimulation also induced an increase of [Ca2+]i in the touched cell, and this increase of [Ca2+]i spread to the surrounding cells. The [Ca2+]i signal travelled a distance of 100-200 microns within 20-40 s and then diminished. The presence of cell-to-cell communication between adjacent mammary cells through gap junction was indicated by injection of lucifer yellow and measurements of electrical coupling (coupling constant = 0.2-0.3). The mechanically induced increase of the [Ca2+]i signal spread to adjacent cells even when the stimulated cell had no physical contact with them. In the absence of fluid movement, the pattern of the spread of the [Ca2+]i signal was a concentric circle. However, in the presence of fluid movement, the pattern changed to elongate to the direction of the flow. These findings suggested that a certain factor was released from the mechanically stimulated cell to the extracellular space, and this factor induces the increase of [Ca2+]i in surrounding cells.     

Developmental Sequence and Intracellular Sites of Synthesis of Three Structural Protein Antigens of Influenza A2 Virus

Specific antisera for hemagglutinin (HA) and neuraminidase antigens of influenza A(2) virus (A(2)E) were produced through the segregation of the two proteins in reciprocal viral recombinants of A(2)E and A(0)e viruses. Gamma globulin fractions of these specific antisera and of antiserum specific for the nucleoprotein (NP) antigen of A(0)e virus were conjugated with fluorescein isothiocyanate and employed to follow the synthesis of the three structural proteins in clone 1-5C-4 human aneuploid cells, with parallel measurement of serological and biological activity of the antigens by other techniques. In this system, NP antigen appeared first (at 3 hr) in the cell nucleus, whereas HA and neuraminidase appeared coincidentally, at 4 hr after infection, in the cytoplasm. The initial detectability of biological or complement-fixing activity of the proteins coincided with their demonstrability as stainable antigens. Late in infection, all three antigens were detected at the cell surface. Antibody specific for HA partially blocked the intracellular staining of neuraminidase and inhibited the enzymatic activity of both extracted and intact extracellular virus. These observations suggest the close intracytoplasmic proximity of the two envelope antigens and perhaps their initial association in a larger protein.     

Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion

The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. We reported previously that E-cadherin-alpha-catenin fusion molecules showed cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Here, we constructed deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay revealed that the fusion molecules containing the ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifted the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not completely support the strong state of cell adhesion activity. Our studies showed that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity.     

Isolation and Classification of Temperature-Sensitive Mutants of Influenza B Virus

We isolated 25 temperature-sensitive mutants of B/Kanagawa/73 strain generated by mutagenesis with 5-fluorouracil and classified them into seven recombination groups by pair-wise crosses. All mutants showed a ratio of plaquing efficiency at the nonpermissive temperature (37.5 C) to the permissive temperature (32 C) of 10^–4 or less. At 37.5 C most of group I, II, and III mutants did not produce appreciable amounts of protein, but all other group mutants were protein synthesis-positive. A group VII mutant produced active hemagglutinin (HA) and neuraminidase (NA) at the nonpermissive temperature, but Group V mutants produced only active NA and were defective in the HA molecule. The other group mutants, including group IV mutants with mutation only in the NA gene (8, 10), lacked both activities at the nonpermissive temperature. One of nine influenza B virus isolates in 1989 had EOP 37.5/32 of 1/3 × 10^–2 and belonged to recombination group VII.     

Ultrastructural study on the stylar transmitting tissue in Japanese pear

Summary The stylar transmitting tissue in the mature pistil of the Japanese pear consists of its component cells and intercellular heterogeneous secretions. The cytoplasm of the periplasmic region contains two different organelles that are characteristic of floral bud development. One of these is the vesicle, which is derived from rough ER and transferred to the periplasmic region of the cell during an early stage of the floral bud. The other one is the lipid droplet, which reacts to polysaccharidic staining and is seen throughout floral bud development. The lipid droplets are closely associated with the Golgi bodies and seem to be dissolved in the vacuole. The materials found in the vacuoles appear to diffuse and pass through the cell walls as intercellular substances.     

Maternal effects on progeny body size and color in the desert locust, Schistocerca gregaria: examination of a current view

Hatchling body color and size of the desert locust, Schistocerca gregaria, are determined by the population density of the mothers during their reproductive period. Smaller green hatchlings are produced by adults at low population density (solitarious conditions) and larger dark hatchlings at high population density (gregarious conditions). One claim states that a pheromonal factor secreted by gregarious mothers into foam plugs of egg pods induces darkening in hatchlings. Previous research suggests that the foam factor can be removed by separating eggs individually within 1h of deposition, causing presumptive gregarious eggs to hatch without darkening. The present study re-examined this claim and possible factors that have been proposed which could account for the difference between our results and those reported earlier. Early separation was performed on eggs with a low mortality rate. The results showed that the egg separation did not increase the incidence of green hatchlings. Once chorionated in the ovary, eggs remained unchanged in size until the second day after oviposition in either isolated or crowded locusts. This and other results suggest that the phase-dependent differences in body size and color of hatchlings are established in the ovary and that modifications by the accessory gland factor either in the oviduct or after deposition are unlikely.     

Maternal effects on progeny size, number and body color in the desert locust, Schistocerca gregaria: Density- and reproductive cycle-dependent variation

The effects of rearing density and maternal age on the progeny size, number and coloration of the desert locust, Schistocerca gregaria, were investigated. Isolated-reared females deposited smaller, but more eggs than crowd-reared females. The former produced smaller and more eggs with age, whereas the latter showed a tendency to produce larger and fewer eggs over time. A similar tendency was also observed with virgin females, indicating that mating or the presence of males was not important. The first egg pod produced by each mated crowd-reared female contained significantly smaller and more eggs than did the subsequent egg pods. The former often produced many green hatchlings (0-100%) characteristic of solitarious forms, whereas the egg pods deposited after the first pod produced predominantly black hatchlings typical of gregarious forms. Adults were highly sensitive to a shift in rearing density and quickly modified the quality and quantity of their progeny depending on the density encountered. The number of eggs per pod was influenced not only by the mother's rearing density but also by rearing density of the grandmother. The present results demonstrated that the characteristics of progeny are influenced not only by the crowding conditions experienced by the mother and grandmother but also by the mother's reproductive cycle.