In vitro stimulation of cell death in the moulting glands of Oncopeltus fasciatus by 20-hydroxyecdysone

The moulting glands of the milkweed bug, Oncopeltus fasciatus, normally degenerate just before the time of ecdysis to an adult (day 7 of the fifth instar). Morphologically normal cell death can be prematurely stimulated in vitro by 20-hydroxyecdysone. Breakdown is triggered by a 24-hr period of exposure to 20-hydroxyecdysone, but an additional incubation period is required before clear signs of degeneration are manifested. Glands removed after the onset of endogenous ecdysteroid secretion degenerate in vitro in the absence of added hormones. Thus, in the moulting glands of Oncopeltus, ecdysteroids appear to act as an important trigger for metamorphic cell death.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

The lambda head-tail joining reaction: purification, properties and structure of biologically active heads and tails

Procedures were developed to obtain biologically active lambda heads and tails at high purity with 20 to 40% recovery. Free heads, free tails and phage particles differ markedly in stability. Phage are stable in solutions containing Mg²⁺ but tails are not. The protein subunits which form the shaft of the tail dissociate in the presence of Mg²⁺ and form multisubunit spherical structures. EDTA protects free tails against inactivation but disrupts heads and phage particles. The four carbon diamine, putrescine, stabilizes heads against inactivation; the three and five carbon diamines are less effective. Electron micrographs reveal a new “knob” structure at the distal end of the tail fiber of phage and of free tails. Tails released from EDTA-disrupted phage possess a “head-tail connector”, a structure not present on the tail before its joining with a head.     

Effects of precocene I and II on flight behaviour in Oncopeltus fasciatus, the migratory milkweed bug

Treatment of newly emerged adult Oncopeltus fasciatus with the corpus allatum inhibitors. 7-methoxy-2,2-dimethyl chromene or 6,7-dimethoxy-2,2-dimethyl chromene (preocene I and II) results in an inhibition of long-term flight (presumed migratory) behaviour in both males and females and inhibition of oögenesis in females. Treatment of reproductive females with precocene briefly stimulates flight behaviour (which ceases in such females as oviposition begins) and then subsequently inhibits it. Oviposition also ceases in such females and oöcyte resorption occurs. Topical application or injections of JH III to precocenetreated animals results in immediate restoration of the tendency to make long tethered flights while corpora cardiaca extract injections, corpora cardiaca implantation, sham implantation, sham injections and sham topical applications were ineffective in restoring prolonged flight behaviour to precocene-treated animals.Restoration of flight by JH III injection was observed within 1 hr after treatment with the hormone. These results indicate that JH is necessary for prolonged flight and presumably migratory behaviour in this species and its stimulatory effect on flight behaviour is immediate. Possible mechanisms of action of the hormone on flight behaviour are discussed.     

Maintenance of the shore-level size gradient in the marine snail Tegula funebralis (A. Adams): Importance of behavioral responses to light and sea star predators

The intertidal gastropod, Tegula funebralis (A. Adams) exhibits a shore-level size gradient with mean shell size increasing in a down-shore direction. Snails transferred to zones where they do not usually occur migrated back towards their original zone, thus re-establishing a size gradient and implying differential movement among size classes. Both large (≥2.1 cm shell width) and small (≤ 1.77 cm) snails were photonegative on a horizontal surface and geonegative in the laboratory; there were no statistical differences between size classes. Light, however, inhibited upward, or caused downward, movement of large snails on vertical surfaces. Small snails were unaffected, ranging higher on illuminated vertical surfaces than large snails. Both sizes exhibited similar distributions in the dark. In an experimental chamber providing both emersed and immersed surfaces, T. funebralis established vertical size gradients when the chamber was illuminated from above. It is suggested that light is an important factor in the formation and maintenance of Tegula's shore-level size gradient.In response to water-borne chemicals derived from the sea star Pisaster ochraceus (Brandt), large snails moved up vertical surfaces in greater proportion than small. In response to contact with the predator, large snails moved away faster than small and individuals collected from crevices in the field moved away slower than those collected from open rock faces. Although predation may select for a size gradient in Tegulafunebralis, it is unlikely that responses to predatory sea stars directly and proximally cause or maintain them over the short term.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

The effects of neonatal 6-hydroxydopamine induced sympathectomy on response inhibition in extinction.

Superfused helical strips of canine anterior mesenteric arteries and veins and canine dorsal metatarsal veins contract in response to prostaglandin B₂ (PGB₂). Reserpine pretreatment and phentolamine reduce the constrictor response to PGB₂. PGB₂ enhances the contractile responses of these preparations to potassium, barium and norepinephrine. PGB₂ also produced a shift to the left in the duration of the barium response curve. The data presented demonstrate that PGB₂ is not an inactive metabolite of PGA₂ metabolism but possesses potent constrictor activity probably dependent on release of norepinephrine from adrenergic nerves. Furthermore, PGB₂ enhances the responses of vascular smooth muscle to vasoactive stimuli.     

Sound localization by the Hawaiian squirrelfishes, Myripristis berndti and M. argyromus

Sound localization was investigated in a large pond open to a bay and similar to the normal environment of the animals. Observations were made of fish movements towards one of two underwater loud-speakers emitting squirrelfish alarm calls normally produced in response to predators. When the sound source was within 2·0 m of the test cage housing the fish, the subjects faced and moved toward the speaker. The animals responded some of the time when the source was within 3·0 m but generally did not orient to the sound source when the speaker was beyond 3·0 m. Response loss was correlated with the fish being in the acoustic far-field. Possible cues which release and direct localization remain unknown, but include particle velocity information alone, or some change in particle velocity: pressure relationships.     

The allomerization of chlorophylls a and b with superoxide anion

The interaction of chlorophylls a and b with electrochemically prepared superoxide anion was studied in aprotic solvent. It was found that O₂^?·causes the deprotonation at carbon C-10 of ring V and production of chlorophyll enolate ions. The intermediate anions undergo rapid oxidation into corresponding chlorins. Pyrochlorophyll a, which lacks the C-10 carboxymethyl group, did not show the transformation. It is suggested that more strong free radical oxidants (e.g., HO₂·, or RO₂·) are capable of abstracting the hydrogen atom at C-10. The possible significance of free radical deprotonation and oxidation in chlorophyll allomerization is discussed.     

Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma (COLO 316)

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous desmosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.     

Hyperpigmented patches in the skin of the newt Notophthalmus viridescens

In the integument of the red-spotted newt there occasionally appear patches of skin which are at the same time melanistic and iridescent. Such hyperpigmented patches have been found on the back, on the tail and on the dorsal surface of both fore and hind limbs. Cytological examination of several such areas revealed the presence of large numbers of chromatophores distributed throughout the dermis. The majority of the chromatophores consisted of atypically large and dendritic melanophores, which contained typical pigment granules. The iridescence resulted from a high incidence of iridophores. Xanthophores also were found in considerable abundance. This extensive and apparently random intermingling of melanophores, iridophores and xanthophores in limited areas constitutes a striking exception to the usual distributional patterns of pigment cells in this animal.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

The isotype cycle: successive changes in surface immunoglobulin classes expressed by the antigen-binding B-cell population during the primary in vivo response

Using the antigen-binding inhibition method, capable of revealing any combination of three surface Ig (sIg) isotypes on a population of antigen-binding cells (ABC) (S. Kanowith-Klein, E. S. Vitetta E.L. Korn, and R.F. Ashman, J. Immunol.122, 2349, 1979) we have defined the sequence of antigen-induced changes in the expression of sIgM, sIgD, and sIgG on the sheep erythrocyte (SRC) antigen-binding B-cell population (SRC-ABC) throughout the in vivo primary immune response. The majority of nonimmune B-ABC simultaneously expressed M and D (M⁺D⁺G^?). By Day 3 sIgG had appeared, mainly on cells already bearing sIgM and sIgD. By Day 5, other G⁺ populations appeared: M⁺D^?G⁺ and M^?D^?G⁺. By Day 12, M⁺D^?G⁺ ABC declined, while M^?D^?G⁺ ABC remained predominant for another month. By 6 months, the sIg phenotypes on the ABC had returned to the original nonimmune pattern, mainly M⁺D⁺G^?; but the absolute number of 6-month immune ABC was four times greater than that of nonimmune ABC. This cyclical change in sIg expression was confined to the B-cell population expressing receptors specific for the immunizing antigen, and affected the large majority of such cells. Twelve days after immunization with SRC, ABC specific for a non-cross-reacting antigen still mainly expressed the nonimmune sIg phenotype, M⁺D⁺G^?.     

Regulation of insect prothoracic glands: Existence of a haemolymph stimulatory factor in Manduca sexta

The primary regulator of ecdysone biosynthesis by insect prothoracic glands is the prothoracicotropic hormone. However, it now appears that other factors, secondary regulators, may modulate prothoracic gland activity. One such factor has been isolated from the haemolymph of Manduca larvae. This haemolymph factor stimulates in vitro ecdysone synthesis by larval and pupal prothoracic glands by approx. 5-fold. It has an apparent mol. wt of ～330 kD, is protease-sensitive and is heat labile, the latter clearly distinguishing it from the prothoracicotropic hormone. Further, its steroidogenic effects and those of prothoracicotropic hormone are additive. Treatment of larval or pupal prothoracic glands with both moieties simultaneously effects an approx. 10-fold increase in ecdysone synthesis. The haemolymph titre of the stimulatory factor is low at commitment of the last-larval instar, then increases by approx. 3-fold later in the instar during pharate-pupal development. This increase in the titre is sufficient to effect a significant increase in prothoracic gland activity that could be physiologically important. Thus, it appears that the fluctuating level of this haemolymph stimulatory factor may act in conjunction with prothoracicotropic hormone to regulate the haemolymph ecdysteroid titre by modulating the ecdysone biosynthetic activity of the prothoracic glands.     

Sodium gradient-stimulated transport of L-carnitine into renal brush border membrane vesicles: kinetics, specificity, and regulation by dietary carnitine

L-Carnitine transport by rat renal brush border membrane vesicles was stimulated by a Na+ gradient (extravesicular greater than intravesicular). Total carnitine entry was 2.7 and 3.2 times higher at 15 S in the presence of a 100 mM NaCl gradient than when the vesicles were incubated isoosmotically in buffered 100 mM KCl or buffered mannitol, respectively. Specific carnitine transport (total entry minus contribution from diffusion) was stimulated 3.6- and 5.7-fold, respectively. An "overshoot" was observed for total carnitine entry in the presence of a Na+ gradient but not in the presence of a K+ gradient or in the absence of an ion gradient. L-Carnitine transport was saturable. KT and Vmax for total carnitine transport were 0.11 mM and 11.6 pmol S-1 mg protein-1, respectively, and for Na+-gradient-dependent carnitine transport, 0.055 mM and 5.09 pmol S-1 mg protein-1, respectively. The transport process was structure-specific for a quaternary nitrogen and carboxyl groups attached by a 4- to 6-carbon chain, but without other charged functional groups. Other evidence for a carrier-mediated process included trans-stimulation of transport by intravesicular carnitine and a peak of activity at near physiological temperature. Kinetic data derived from this study, coupled with data from previous physiological studies from this laboratory, suggests that carnitine transport by the brush border membrane is not limiting for carnitine reabsorption. Dietary carnitine (1% of diet for 10 days) reduced by 52% the rate of carnitine transport across the brush border membrane in vitro, without affecting rates of D-glucose, L-lysine, L-glutamic acid, or L-alanine transport. Down-regulation of carnitine transport may prevent excessive or toxic accumulation of L-carnitine in renal tubular cells exposed to high extracellular carnitine concentrations.     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.     

X-ray-induced mutations affecting the level of the enzyme alcohol dehydrogenase in Drosophila melanogaster: frequency and genetic analysis of null-enzyme mutants.

The frequency of X-ray-induced (null-enzyme) mutations at the alcohol dehydrogenase locus in Drosophila melanogaster was measured. The rate of recovery of chromosomes that fail to direct the synthesis of a functional Adh protein is 3 x 10(-8) per R for chromosomes that do not include large chromosome rearrangements. However, this analysis excludes a larger number of chromosomes that are "null-enzyme mutations" because thye are deleted for the region of the Adh locus. The dose of X-rays required to induce a frequency of non-deletion null-enzyme mutants equal to the spontaneous frequency is about 73 rad calculated from the data reported in this communication.     

Biogenic magnetite as a basis for magnetic field detection in animals

Bacteria, sharks, honey bees, and homing pigeons as well as other organisms seem to detect the direction of the earth's magnetic field. Indirect but reproducible evidence suggests that the bees and birds can also respond to very minute changes in its intensity. The mechanisms behind this sensitivity are not known. Naturally magnetic, biologically precipitated magnetite (Fe₃O₄) has been found in chitons, magnetotactic bacteria, honey bees, homing pigeons, and dolphins. Its mineralization in localized areas may be associated with the ability of these animals to respond to the direction and intensity of the earth's magnetic field. The presence of large numbers (～10⁸) of superparamagnetic magnetite crystals in honey bees and similar numbers of single-domain magnetite grains in pigeons suggests that there may be at least two basic types of ferrimagnetic magnetoreceptive organelles. Theoretical calculations show that ferrimagnetic organs using either type of grain when integrated by the nervous system are capable of accounting for even the most extreme magnetic field sensitivities reported. Indirect evidence suggests that organic magnetite may be a common biological component, and may account for the results of numerous high field and electromagnetic experiments on animals.     

Kinetics of the reaction between hydrogen selenide ion and oxygen

Hydrogen selenide ion (HSe^?) reacts with oxygen in the following manner: HSe^? + 1/2O₂ → Se^o + OH^?. Interest in the kinetics of this reaction comes from the fact that selenide is an important product in the metabolism of the essential trace element selenium. Using polarography to monitor both selenide and oxygen, we have found the reaction exhibits complex kinetics, including autoaccelerating behavior and the generation of reactive intermediates capable of inducing reactions in other substances present. Probable intermediate species include superoxide, peroxide and polyselenides. The reaction is slow with respect to diffusion controlled reactions, but fast with respect to the time required to prepare solutions for biological study. Selenide concentrations greater than 10^?6 M decay to give solutions of predominantly colloidal elemental selenium less than 3 minutes after exposure to atmospheric levels of oxygen.     

The cell cycle and its relationship to development in Dictyostelium discoideum.

When deprived of exogenous nutrients some amoebas of Dictyostelium discoideum do continue to progress through the cell cycle. There are two distinct periods when mitotic cell division occurs. Labeling studies show that during the first period, which begins at the onset of development and ceases at the first visible signs of aggregation (rippling), only those cells which are beyond a certain point in G2 at the initiation of development divide. The second period of mitotic activity begins at tip formation, reaches maximum activity at the grex stage, and ceases during early culmination. Significantly, examination of the development of amoebas harvested when in the stationary phase of growth (and thus arrested in G2) shows that these cells still undergo mitotic cell division during the second period but do not show any such division during the preaggregation phase. The extent to which increases in cell number can be taken to be indicative of mitotic cell division varies from one culture to another due to the presence of variable numbers of multinucleate cells which become mononucleate during the first 10 hr of development. However, when due allowance has been made for the existence of these cells in axenically growing amoebal populations, our data show that by completion of fruiting body construction there has been a doubling in cell number as a direct result of mitotic cell division. Nuclear DNA synthesis also occurs at two distinct periods during development, these coinciding with the periods of mitotic activity. However, since no more than 35% of the cells have undergone nuclear DNA synthesis by the end of the developmental phase, our results are inconsistent with the conclusion that all cells accumulate at a position in G2 at the time of aggregation. Our results do suggest, however, that mitotic cell division of a fraction of the cells may be an integral part of the developmental phase.