DEVELOPMENTAL STUDIES OF ERYTHROCLADIA ROSENVINGE IN CULTURE

Erythrocladia subintegra has been cultured for over 3 years. The life history includes reproduction by monospores, a long period of ameboid motility by monospores, their germination and growth into monostromatic disk-shaped plantlets composed of adherent, often dichotomously branched filaments. Plants become centrally polystromatic with age and produce monospores. Variations in plant size and shape, cell size and shape, spore size, shape, and motility are discussed. Variations in plants of E. subintegra grown on different substrates have been studied and discussed in relation to other species in the genus.     

The complex for replication initiation of<Emphasis Type="Italic">Escherichia coli</Emphasis>

We probed the complex betweenoriC and DnaA protein using two types of mutants inoriC. Base changes in the DnaA binding sites, DnaA boxes, had little effect on origin function. Mutations which change the distance between DnaA boxes R3 and R4, on the other hand, inactivatedoriC unless the mutation deleted or inserted one complete helical turn. Origins with other 10 base pair insertions in the interval between DnaA boxes R2 and R3 were functional, but not insertions in the R1–R2 interval. FIS protein binds to a bipartite site inoriC between DnaA boxes R2 and R3. A model for theoriC/DnaA complex based on these results suggests an array of DnaA monomers with a 34 Å spacing upon whichoriC is arranged.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Effect of Tetrahydrobiopterin on Serotonin Synthesis, Release, and Metabolism in Superfused Hippocampal Slices

The effects of 6R-5,6,7,8-tetrahydro-L-biopterin (6R-BH4), the in vivo cofactor for tryptophan hydroxylase, on the synthesis, release, and metabolism of serotonin were studied in superfused slices from rat hippocampus. 6R-BH4 did not alter the spontaneous release of [3H]serotonin but it did significantly increase release when slices were depolarized with 30 mM KCl. Under the same incubation conditions, 6R-BH4 altered neither the synthesis (basal or tryptophan-stimulated) nor the metabolism of serotonin in hippocampal slices. The synthetic pteridine 6-methyl-5,6,7,8-tetrahydropterin also augmented release under depolarizing conditions whereas biopterin, the oxidized form of 6R-BH4, did not. The 6S isomer of BH4, which is relatively inactive as a cofactor for tryptophan hydroxylase, was equipotent with 6R-BH4 in stimulating serotonin release. 6R-BH4 did not inhibit serotonin uptake nor did it function as a serotonin autoreceptor antagonist to increase release. A direct serotonin releasing effect of 6R-BH4, like that produced by p-chloroamphetamine, could also be ruled out. At suboptimal concentrations of extracellular calcium, the KCl-induced release of 3H was significantly reduced, yet the increase in release caused by BH4 remained the same in magnitude. It is concluded that 6R-BH4 increases the depolarization-induced release of serotonin through an interaction with the release mechanism itself, possibly by enhancing calcium influx or by increasing the sensitivity of the release mechanism to calcium. The effects of 6R-BH4 on serotonin release are independent from its function as the cofactor for tryptophan hydroxylase.     

A collaborative study to establish a U.S. reference for tissue plasminogen activator (t-PA)

In 1987 the Second International Standard for tissue plasminogen activator (t-PA) was established by the World Health Organization following an international collaborative study. At that time, the Center for Biologics Evaluation and Research (CBER) decided to establish a national reference t-PA to be used in lot release potency testing of Alteplase, a licensed t-PA biological or of other t-PAs in development. A candidate recombinant t-PA (rt-PA) preparation was donated by Genentech, Inc. (South San Francisco, California) for this purpose and a collaborative study was launched to calibrate this material against the 2nd I.S. Four laboratories (including the Center for Biologics Evaluation and Research (CBER) and three manufacturers) participated in the study to establish the potency of the rt-PA preparation using a clot lysis assay. The results indicate that the potency of the U.S. reference for t-PA is 2900 international units (IU) per vial.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.     

Author index to volume 167

The N-terminal amino acid sequences of two chloroform/methanol soluble globulins from barley and one form wheat are reported. They are homologous with N-terminal sequences previously reported for α-amylase and trypsin inhibitors from cereals and 2 S storage proteins from castor bean and rape. Three albumins were also purified from Aegilops squarrosa and Triticum monococcum. These had N-terminal amino acid sequences most closely related to the α-amylase and trypsin inhibitors. The relationships of this superfamily of seed proteins are discussed.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

The cellular basis of wing size modification in Drosophila: The effects of the miniature gene,crowding, and temperature

To elucidate the mechanisms whereby genes and environment influence wing size, we investigated the effects of various rearing temperatures and larval crowding conditions on the wings of the mutant miniature and wild-type fruit flies. In adults we monitored wing size, cell number, wing thickness, cell density; in larval imaginal discs we looked for cell death. Cell density was inversely proportional to wing size. Of particular interest was the finding that smaller wings tend to be thicker. Electron microscope studies showed that the miniature wing layers are grossly abnormal. We hypothesize that these abnormalities are due to abnormal cell flattening of the wing epithelial cells, and we conclude that gene and environmental effects on cell flattening may be an important component in determining cell density and hence organ size.