Effects of protease inhibitors on sperm-related events in sea urchin fertilization

Evidence for sperm-borne proteolytic enzymes exposed during the acrosome reaction in sea urchin sperm has been accumulating. To investigate the possible role(s) such enzymes have in fertilization, we studied the effects of several protease inhibitors on sperm-related events. Soybean trypsin inhibitor, Nα-p-tosyl-l-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, and chymostatin neither reduced the number of acrosome reactions nor interfered with gamete binding. p-Nitrophenyl-p′-guanidinobenzoate caused sperm to fuse into irregular clumps, rendering them unable to fertilize eggs. However, l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin, prevented the acrosome reaction in Strongylocentrotus purpuratus, S. droebachiensis, and Lytechinus pictus. The effects of TPCK on sperm in subsequent steps of fertilization were also investigated. First, gamete binding assays were performed on fixed eggs. This precluded any effects TPCK might have had on egg-derived secretions (e.g., proteases). Binding of prereacted sperm occurred with both fixed and living eggs. However, fertilization of living eggs in the presence of TPCK was greatly reduced, even though sperm had been prereacted with egg jelly. Vitelline coats were then removed from eggs by trypsin treatment. Eggs in TPCK fertilized and developed normally after the above treatment. These observations are consistent with the hypothesis of a sperm protease participating in the acrosome reaction and the penetration of the egg vitelline coat in the sea urchin.     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

Oligo(U) sequences present in sea urchin maternal RNA decrease following fertilization

Oligo(U) tracts were identified and measured in RNA from sea urchin eggs and embryos using a quantitative assay based on the amount of [3H]poly(A) protected from RNase T2 in duplexes with the oligo(U). The oligo(U) amounted to 0.0035% of egg RNA (0.063 X 10(-12) g/egg) and decreased to 0.0015% (0.027 X 10(-12) g/embryo) by 2 hr after fertilization. The oligo(U) tracts had a maximum size of 15-30 nucleotides and were associated with two size classes of RNA. In eggs about half were in 100 to 200 nucleotide RNA and half in mRNA-sized molecules. After fertilization, the oligo(U) in the population of large-mRNA-sized molecules was greatly reduced.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Hemogloblin α-gene duplication in macaques: Individual Macaca nemestrina with three structurally different α chains

The diversity of hemoglobin phenotypes observed among Malaysian Macaca nemestrina (pig-tailed macaques) has been attributed, in part, to the presence of duplicated α-chain loci in some members of this species. To date, evidence in support of this view has been indirect, consisting of variation in proportions of α^I (Asp₇₁, Gln₇₈) and α^II (Asp₇₁, His₇₈) chains among presumptive heterozygotes. However, the discovery that erythrocytes from some M. nemestrina contain α^I and α^II chains in company with a third, or α^III, chain (Gly₇₁, Gln₇₈) provides direct evidence of duplicated α-chain loci.     

Fluorescence properties of a benzo(a)pyrene 7,8 dihydrodiol 9,10-oxide-DNA adduct. Conformation and effects of intermolecular DNA interactions

The pyrene-like fluorescence of the covalent benzo(a)pyrene diol-epoxide-DNA complex prepared by reacting 7,8,-dihydrodiol 9,10-epoxy benzo(a)pyrene (BPDE) with DNA in aqueous solution $\text{in vitro}$, has been investigated. It is shown that this fluorescence is $\text{sensitive}$ to molecular oxygen, to the concentration of $\text{native}$ DNA and to the ionic strength (KCl concentration), but is $\text{insensitive}$ to the concentration of $\text{denatured}$ DNA. These effects are related to the conformation of the pyrene-like chromophore of BPDE. Most of the fluorescence of a $\text{dilute}$ solution of the DNA-bound benzo(a)pyrene derivative originates from binding sites in which the pyrene moiety is not intercalated between the DNA base pairs, but is located on the outside of the DNA double helix.     

Kinetic identification of component X as P430: a primary electron acceptor of Photosystem I

Kinetics of dark recoveries of Component X, Center A, and Center B at 20 and 0 °C after a 30-s illumination were studied in membrane fragments from a blue-green alga by using low temperature electron paramagnetic resonance spectroscopy in combination with a quick-freeze method. These kinetics were compared with those obtained by spectrophotometry under the same conditions. Contrary to the currently popular view, the result strongly suggests that Component X, rather than Center A or Center B, is P430.     

Effect of 5-bromodeoxyuridine on appearance of cell-surface saccharides in organ cultures of embryonic pancreas

Rat pancreatic rudiments from Day 15 embryos cultured for 11 days with 20 μM 5-bromodeoxyuridine (BrdU) show selective suppression of acinar cell cytodifferentiation with production of fluid-filled vacuoles lined by undifferentiated cells. Using a battery of lectin-ferritin conjugates (Con A, RCA I, WGA, SBA, and Ulex lectin) we have shown that the majority of these cells express cell-surface glycoconjugate patterns reminiscent of both protodifferentiated cells from Day 15 rudiments and of adult centroacinar (i.e., duct-like) cells. A smaller population of the undifferentiated cells which contain abundant elements of the rough-surfaced endoplasmic reticulum expresses surface saccharide patterns equivalent to those of acinar cells in Day 19 rudiments. These cells, however, lack zymogen granules characteristic of acinar cells in Day 19 rudiments and are similar morphologically to presumptive acinar cells in Day 17 rudiments. Culture of Day 14 pancreatic rudiments with BrdU leads to growth only of undifferentiated cells with duct-like cell-surface saccharide patterns. We interpret these results to indicate that (1) the differentiation program for the acinar cell plasmalemma is established earlier than that for its intracellular organelles; (2) these two developmental programs are independently regulated; and (3) the progenitor of the acinar cell in the protodifferentiated rudiment may be related to the centroacinar or duct-like cell.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster

The relative DNA content of Drosophila melanogaster imaginal leg disc nuclei during larval growth and pupal and adult differentiation was measured by microspectrophotometry. During the larval proliferative phase there were twice as many nuclei in the 4C class as nuclei in the 2C class. At the end of the third larval instar, the proportion of nuclei with a 4C DNA value increased. By 3 hr after pupariation, during pupal cuticle secretion, 90% of the nuclei were in this class. After pupal apolysis which occurs at 12 hr after pupariation, the 4C to 2C ratio was reversed. The increase in the proportion of nuclei with a 2C value was observed until 24 hr after pupariation when 90% of the nuclei were in this class. We propose that most cells divide at least once between pupal and adult differentiation. All of these changes in the cell cycle were correlated temporally with changes in the ecdysteroid titers that occur during these periods.     

Age-dependent changes in myogenic precursor cell compartment sizes. Evidence for the existence of a stem cell

Individual myogenic cells were isolated from the pectoralis muscles of chick embryos from days 8-14 of embryogenesis. When separately cloned, these cells produced three types of colonies in culture: (1) Positive: all cells in the clone were terminally differentiated muscle cells; (2) negative: no cells in the clone were terminally differentiated muscle; (3) mixed: some cells in the clone were terminally differentiated muscle. Positive clones from all ages tended to contain 2n cells (n = 0, 1, 2, 3, 4). Negative clones were found in all sizes and did not cluster around powers of 2 in cell number. Mixed clones were, by far, the most common type among those clones larger than 24 in cell number. Estimates of cell numbers in embryonic muscle tissue revealed that, while the numbers of cells in all myogenic compartments increased steadily with embryonic age, the number and percentage of precursor cells that produced large mixed clones increased dramatically. Subclones, prepared from populations of cells equivalent to large mixed clones, yielded both small positive and large mixed colonies. This indicated that the precursors to the large mixed clones were also precursors to the smaller positive clones. These observations suggest a model for the myogenic lineage in which there exists a stem cell that can generate, by a series of asymmetric divisions, cohorts of terminally differentiated muscle cells. The model can explain the asynchrony of production of terminally differentiated muscle cells both in vitro and in vivo.     

Reaction of BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) with polycytidylic acid. Substitution of the cytosine ring

BCNU has been reacted with polycytidylic acid and two derivatives of CMP, 3-hydroxyethyl-CMP and 3,N⁴-ethano-CMP, have been identified in the acid hydrolysate of the polymer. Their formation accounts for some of the reaction of BCNU with nucleic acids, and may be related to the mechanism of action of this compound.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.