Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.     

Aspects of the Natural History of the Arboreal Harvestman <Emphasis Type="Italic">Cynorta marginalis</Emphasis> (Opiliones: Cosmetidae) Inhabiting a Costa Rican Forest

Relatively little is known about the natural history of the cosmetid harvestmen that inhabit the forests of Central America. The primary objective of this study was to investigate habitat use among adult Cynorta marginalis. In the field, we established 15 transects (40 m in length) and sampled them repeatedly in the morning (0830–1100 h) and evening (1800–2300 h). During 45 morning and 35 evening surveys, we captured and marked 146 males and 112 females. Only three individuals (all females) were recaptured over the course of the study, indicating that the population size at this field site was relatively large. Heavy rains significantly reduced the surface activity of adults (1.1 individuals per transect during heavy rain vs. 3.7 individuals per transect during light or no rain). Harvestmen most commonly used leaves (65% of captures) or tree trunks (31%) as perches, and rarely occupied branches or the leaf litter. Only 18 adults were observed with leg injuries, with two individuals having damage to multiple legs. Perch height was significantly lower during the evening (110.9 cm at night vs. 155.1 cm during the day). Adults were most commonly found alone (85%), however, we observed several harvestmen in male-female pairs (8%), same sex pairs (4%) and heterosexual groups (3%). Although we did not observe reproductive or aggressive behaviors in the field, we observed intraspecific interactions under laboratory conditions and found that some individuals (15%) engaged in courtship suggesting that the sharing of perches by male-female pairs may be related to reproductive activity.     

THE ORIGIN AND FATE OF MICROBODIES IN THE FAT BODY OF AN INSECT

The structure and life history of insect microbodies are described during the development of the fat body from the 4th to 5th larval molt through the 5th to pupal molt. The mature microbodies are flattened spheres about 1.1 x 0.9 µ, with a depression on one side where a dense mass connects the limiting membrane to the core of coiled tubules. They contain catalase and urate oxidase. The precise synchrony of development of insect cells during the molt/intermolt cycle makes it easy to study the life history of particular organelles. Phases of growth are correlated with the hormonal milieu. Mature 4th stage microbodies decrease in size before ecdysis to the 5th stage when they atrophy at the same time as the new 5th stage generation arises. The 5th stage microbodies form as diverticula of the RER and, grow while confronted by RER cisternae. The mature microbodies decrease in size when the fat body engages in massive larval syntheses. At the end of the 5th larval stage, the microbodies are invested by isolation membranes and destroyed before pupation. There are thus two mechanisms for microbody destruction: atrophy of the 4th stage organelles and isolation with autophagy at the end of the 5th stage.     

The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect

The structure and development of the permanent oenocytes of Calpodes ethlius (Lepidoptera, Hesperiidae) are described. There are three sorts of oenocyte. The permanent oenocytes are arranged ventral to the last two pairs of spiracles on abdominal segments 7 and 8 in four clusters of about 45 cells each. The molt cycle oenocytes are ventral to the other spiracles and only enlarge at molting. The subdermal oenocytes differentiate from the epidermis in large numbers shortly before pupation. The permanent oenocytes are large polyploid cells characterized by a cytoplasm of densely packed smooth tubular endoplasmic reticulum, and a plasma membrane invaginated in a meshwork of tubes ending in a reticular layer about 12 micro below the surface. There are two sorts of Golgi complex, one small and of conventional form, the other composed of clouds of microvesicles. 'Dense bodies', believed to belong to the microbody class of organelles, arise directly from the STER. There is a variety of membranous and 'crystalline' inclusions. The formation of isolation membranes from the tubular endoplasmic reticulum, and the origin of isolation bodies and autophagic vacuoles are described. Some autophagy takes place at all times in the molt/intermolt cycle, but there are phases of massive autophagy before the 4th-5th molt and the 5th-pupal molt. These phases coincide with pinocytosis of blood proteins and overlap with or are followed by phases of nuclear replication, RNA synthesis (ribosomes) and ER regeneration. Nuclear blebbing occurs before pupation. The morphology of the oenocytes is most like that of vertebrate cells engaged in steroid hormone synthesis. It is pointed out that the oenocytes rather than the prothoracic glands could be the source of ecdysone and the stimulus for molting.     

The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma

The Total (Peroxyl) Radical-trapping Antioxidant Parameter (TRAP) of six freshly prepared human plasma samples and 45 frozen plasma samples has been determined. It is shown that contributions from urate (35-65%), plasma proteins (10-50%), ascorbate (0-24%) and vitamin E (5-10%) to TRAP account for all of the peroxyl radical-trapping antioxidant activity in the majority of the samples. The changes in concentrations of the plasma antioxidants during peroxyl radical attack show that the first line of defense is provided by the plasma sulfhydryl groups, even urate being spared during the initial stages of the reaction. The modes of action of all of these plasma antioxidants and possible interactions between them are discussed, with particular emphasis on the abilities of the water-soluble antioxidants to regenerate or spare the only lipid-soluble antioxidant, vitamin E.     

Amphipods, isopods and surface currents: a case for passive dispersal in the Bay of Fundy, Canada

Six species of isopods and 18 species of amphipods were collectedin the neuston of the Bay of Fundy and adjacent waters. Collectionswere made over a grid of stations covering 2.4x10⁴ km² duringthree spring, three summer and two autumn surveys. No isopodsand only five species of amphipods were found in spring surveys.Isopods and amphipods were diverse and plentiful in the neustonin summer and autumn. Dominant isopods were Idotea baltica andI.metallica, and dominant amphipods were Calliopius laeviusculusand Parathemisto gaudichaudi. Amphipods and isopods reach theneuston of the Bay of Fundy in three ways. Idotea metallica,the only euneustonic species present, was probably advectedinto the Bay of Fundy from southern waters in summer, and didnot appear to overwinter in the Bay. Most species, includingI.baltica, were collected with drifting littoral vegetation,and we suggest that transport by surface currents is an importantfactor in dispersal of some shoreline crustaceans. Midwaterplankton, such as Parathemisto gaudichaudi, reached the neustoneither by advection in upwelling waters or by an extension oftheir normal diel vertical distribution.     

Tyrosine storage vacuoles in insect fat body

The watery vacuoles first described from larval insect fat body (Chironomus, Voinov, 1927; Aedes, Wigglesworth, 1942; Rhodnius, Wigglesworth, 1967) have been studied in 4th and 5th stage Calpodes larvae. The vacuoles arise at the beginning (E+6–24 hr) of the 4th stadium from plasma membrane infolds that separate from the cell surface as provacuoles less than 1 μm in diameter. These provacuoles grow and fuse with one another through the intermolt until about half the volume of each fat body cell is occupied by a single, large vacuole. The vacuoles begin to disappear at molting. Their membrane is either incorporated into the plasma membrane by exocytosis or fragmented into vesicles that fuse to become lamellar bodies where the membranes are presumably digested. All the vacuoles have gone by a few hours after ecdysis.The tyrosine content of the fat body increases and decreases in proportion to the size of the vacuoles. As the vacuoles decrease at molting the titre of tyrosine in the hemolymph is transiently elevated at the time when there is most demand for phenolics for cuticle stabilization. Crystals having the form of tyrosine crystallize out from vacuoles separated from the fat body. In fat body extracts separated by thin layer chromatography, similar crystals occur only in the eluates from spots corresponding to tyrosine. The vacuoles are therefore presumed to be tyrosine stores used in cuticle stabilization at molting. They correspond to a type of aqueous storage compartment that is well known in plants but hitherto little recognized in animal cells.