Maturation-related changes in mass and elemental contents of secretory granules as measured by electron-microprobe

Summary The relationship between granule density, protein content, and Ca and S contents were studied in two secretory granule fractions, from parotid glands of the rat, previously shown to constitute different stages in granule maturation. The density of the lighter fraction was between 1.133 and 1.142 g/ml, while that of the heavier fraction was greater than 1.142 g/ml. The mean protein content of the denser granules was 12% greater than that of the lighter granules (P<0.03), while the dry-mass elemental concentrations in the two granule fractions were unchanged. These results indicate that protein is added to granules during the maturation process (presumably by vesicular traffic), and that the resulting increase in granule density is not driven simply by decrease in water content and/or increased concentrations of inorganic Ca or S in the granules. The elemental concentration values also indicate that the diffusible elements permeate the granule membrane during the fractionation procedures.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

Effects of D2O on the movement of chromosomes and the shortening of kinetochore spindle fibers in anaphase in dividing spermatocytes of the grasshopper, Mongolotettix japonicus

Spindles in anaphase of dividing primary spermatocytes of the grasshopper, Mongolotettix japonicus, were examined with a sensitive polarizing microscope combined with a time-lapse video recorder and a cinematographic apparatus. The pole-to-pole distance of the meiotic spindles was increased and the kinetochore fibers were more birefringent in the presence of 40% D2O. However, the rate of shortening of the kinetochore fibers in anaphase was not affected by D2O. This indicates that D2O did not inhibit microtubule disassembly in anaphase, supporting the earlier observations (3, 18) that D2O did not "stabilize" the spindle microtubules at concentrations below 45%. We confirmed that D2O, at the concentration mentioned above, neither promotes nor inhibits the anaphase A. However, the overall sequence of anaphase was considerably extended in the presence of D2O, presumably due to the increased pole-to-pole distance.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Is there an energy conservation “system” in brain that protects against the consequences of energy depletion?

A poorly understood marked decrease (circa 50% of control) in local cerebral glucose utilization is caused by sublethal doses of NaCN. The decrease is global, occurring in essentially all brain regions and is entirely reversible within hours, leaving no obvious pathology. This event is not unique to NaCN in so far as a strikingly similar pattern of decreased glucose utilization occus with some other toxins. Nor can it be attributed to a direct action of NaCN since local application by microdialysis to the striatum produces a global depression. These results imply that some widely distributed system or substance is involved. We speculate the existence of a system possibly related to the reticular activating system that senses a fall in energy production and acts globally to make cells quiescent and thus would give some protection from excitotoxic driven damage.Special issue dedicated to Dr. Sidney Ochs.     

Visual prey discrimination of queen and worker ants by a generalist lizard

Laboratory experiments were conducted to test the hypothesis that widelyforaging generalist lizard,Eumeces okadae, visually discriminates palatable queen ants from unpalatable worker ants. The workers ofCamponotus japonicus andLasius niger were rejected on sight, while the queens of both species were eaten with little prior chemical examination by tongue flicks or licks. Comparison of the lizards' responses towards the workers and wingless queens of similar size indicated that neither body size nor presence or absence of wings accounted for difference in responses toward the 2 ant castes. The lizards probably discriminated different ant castes by body proportions.     

Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit-shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10^?8 joules per μm² was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time-lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half-bivalent with the partner half-bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.     

REPRODUCTIVE ISOLATION BY HOST SPECIFICITY IN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES

Crossing experiments and food-choice tests show that two sympatric species of phytophagous ladybird beetles, Epilachna niponica and E. yasutomii, are reproductively isolated by host-plant specificity. Adult beetles selected their natural hosts when given choices, though some accepted the host of the other species when no choice was offered. In each species, survival of larvae to the second instar was significantly higher on their own host plant. No evidence for sexual isolation, gametic isolation, hybrid inviability, or reduced hybrid fertility was detected. Reproductive isolation by host specificity is an important prerequisite for certain models of sympatric speciation. Although the present example supports the plausibility of such models, an allopatric origin of host-plant specificity cannot be discounted.