Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes

A voltage-gated hydrogen ion-selective conductance has been previously described in the immature oocyte of the urodele amphibian Ambystoma. The present study was prompted by reports that changes in membrane voltage and internal pH, as well as in internal sodium ion concentration, occur during the hormone-induced maturation of oocytes from other amphibians. As activation of membrane currents might mediate changes in internal ion concentrations in addition to altering the membrane voltage, microelectrode recording techniques have been employed to examine changes in membrane conductances which occur during maturation of Ambystoma oocytes. It was observed that during the first 5 hr of maturation the magnitude of the hydrogen ion conductance gradually decreased, and that subsequently there was an increase in the amplitude of a voltage-dependent noninactivating sodium conductance. After 6 to 7 hr, after the loss of the hydrogen conductance and at about the time of germinal vesicle breakdown, the resting potential of the oocyte spontaneously shifted from approximately -10 mV to approximately +30 mV, where it remained until at least 24 hr after the initiation of maturation. This voltage transition was due to the appearance of mechanisms generating inward current in the oocyte membrane; part of this inward current was due to the tonic activation of the sodium conductance. Changes in internal pH and internal sodium ion concentration occurred during maturation, as judged from shifts in the reversal potentials of both hydrogen and sodium currents. A gradual decrease in internal hydrogen ion concentration was observed up until the time of disappearance of the hydrogen conductance (change in internal pH from about 7.15 in immature oocytes to about 7.40 by 3 hr after application of progesterone). This was followed, as sodium conductance increased, by an apparent rise in the internal sodium ion concentration (from about 6 mM to about 17 mM by 10 hr postprogesterone).     

Metals in biochemistry : P.M. Harrison and R.J. Hoare,Chapman & Hall,New York, 1980, 80 pp. $5.95 (paper)

An ultrafiltration technique was used to study stripping by glycine of the first copper and zinc ion equivalents bound by bovine, dog, and rat serum albumins at pH 7.5. Affinity of dog serum albumin for copper was poorer than for the other albumins, consistent with the absence in the former albumin of the copper binding site present at the amino terminus of the latter albumins. Affinities of all three proteins for zinc were similar, suggesting that the albumin amino terminus is not the primary zinc ion binding site.     

Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid)

Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis

The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg²⁺, but not Ca²⁺, is required for adhesion formation, whereas programming for lysis is strongly Ca²⁺ dependent. We have previously reported that the transition metal manganese can substitute for Mg²⁺ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn²⁺ on cytolysis can be reduced by increasing the concentration of Ca²⁺. Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn²⁺ from the Ca²⁺-dependent step. These results suggest that Mn²⁺ blocks a Ca²⁺-dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca²⁺ in programming for lysis is via a Ca²⁺ influx (as a “second messenger?”) or whether Ca²⁺ simply serves as a cofactor at the cell exterior.     

The exchange hypothesis for the formation of chromatid aberrations: an experimental test using bleomycin

The association between bleomycin-induced chromatid aberrations and BUdR-label exchange between sister chromatids was investigated in order to evaluate Revell's exchange hypothesis for the formation of chromatid aberrations. The results of this study indicate that a larger than expected proportion of chromatid breaks can be accounted for by the exchange hypothesis though not all breaks are the result of incomplete exchange.     

Morphogenesis of bacteriophage lambda deletion mutants. II. Escherichia coli mutants which prevent maturation of short genomes.

We have isolated mutants of Escherichia coli which severely reduce the growth of bacteriophage lambda carrying the b221 deletion. Some of the bacterial strains also cause a moderate reduction in the growth of wild-type phage. In the mutant hosts tested, the growth of λb221 is restored by chromosomal alterations producing a non-specific increase in genome length. Thus the defect in growth can be attributed to the physical size of the genome, rather than a genetic effect of the b221 deletion. Our experiments show that the failure to grow results from a block to head morphogenesis and that growth can be restored by mutations in at least two phage head genes. In the accompanying paper we have shown that even in the normal bacterium, the process of packing and cutting the λb221 genome is perturbed as a result of its small size. The block to morphogenesis in the bacterial mutant we have studied most extensively appears to result from an enhancement of the same effect. The experiments described support the hypothesis that there is host participation in the cutting of encapsulated lambda DNA, although it is not yet clear if this involves the direct participation of a host gene product.     

Environmental and chemical dissociation of antibody-dependent phagocytosis from lysis mediated by macrophages: Stimulation of lysis by sulfhydryl-blocking and esterase-inhibiting agents and depression by trypan blue and trypsin

Peritoneal exudate cells and RAW264 macrophage tumor culture line were tested for antibody-dependent effector activity to sheep red blood cells (RBC). Assay in tissue culture dishes showed mostly lysis of targets while tubes promoted phagocytosis. The kinetics suggested that these were independent processes. At concentrations inhibiting ingestion, sulfhydryl-blocking agents iodoacetate, N-ethylmaleimide, and p-chloromercuribenzoate, and esterase inhibitors tosyl-lysine chloromethyl ketone, and diisopropyl fluorophosphate stimulated lysis of RBC. Pretreatment of effector cells but not targets also augmented the lytic reaction. Three other macrophage cell lines with very weak killing activity were stimulated by iodoacetate, but two lymphoid cell lines showed no cytotoxicity with or without the drug. In contrast to these enzyme inhibitors, trypan blue blocked peritoneal exudate and cell line lysis with no effect on phagocytosis. Trypsin pretreatment also inhibited RAW264 but not peritoneal cell cytotoxicity. There was little effect of these agents on macrophage Fc receptors as measured by EA rosettes, or on cell viability or production of lysozyme and β-glucuronidase. We conclude that the two macrophage effector mechanisms are independent, can be inversely modulated by environmental conditions (assay vessel), and are regulated by enzymes or proteins specific for each process.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Structure of lipoprotein (a) studied by spin-labeling

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate causes a 2-fold increase in 1,2-diacylglycerol levels within 15–30 min in cultured chick embryo differentiated myoblasts. The weak tumor promoter 12-O-decanoylphorbol 13-acetate was 250 times less effective and the non-promoter 4-α-phorbol 12,13 didecanoate was ineffective at producing this response. During subcellular fractionation, the stimulated portion of the diacylglycerol was distributed similarly to the plasma membrane fraction. Evidence is presented that this diacylglycerol originates from pre-existing lipid rather than from $\text{de}\text{novo}$ synthesis. Possible implications of these findings with regard to the inhibition of myoblast fusion by the tumor promoter are discussed.     

A model study of ergot alkaloid biosynthesis

A hypothesis of the mechanistic mode of prenylation of tryptophan, an early phase of ergot alkaloid biosynthesis, occurring by way of 3-(α,α-dimethylally)indolenine and its ring-tautomer indoline intermediates and a Cope rearrangement of the latter has been tested in a model study. 3-(α,α-Dimethylallyl)indolines derived from tetrahydrocarbazole and its cyclopentano equivalent have been synthesized by a five-step procedure. The compounds are stable up to ca. 200°C and liberate no benz-prenylated products even on pyrolysis beyond this temperature, thus diminishing the viability of the above biosynthetic hypothesis. The chemistry of the thermolyses is described.     

An extended series of fluorescent sulfhydryl reagents useful in energy transfer measurements

Synthesis of four new fluorescent sulfhydryl reagents is described. All are isomers of the previously synthesized N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonic acid (1,5-I-AEDANS) and its 1,8-isomer (1,8-I-AEDANS). Three of these new probes (1,4-Br-AEDANS, 2,8-Br-AEDANS, and 2,6-I-AEDANS) carry a single sulfonic acid residue and the fourth (3-(2,7)-Br-AEDANS) carries two sulfonic acid residues. The excitation and emission spectrum of each of these probes is distinct when covalently attached to bovine serum albumin. In addition, they all show a single fluorescent lifetime in the range of 8.0 to 20.8 nsec. This extended range of fluorescent isomers can thus be useful for selecting approprlate energy donors in fluorescence energy transfer experiments.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

Interaction of hemopexin with water-soluble porphyrins.

Polyacrylamide-gel electrophoresis and filtration on Bio-Gel P-10 indicate that rabbit hemopexin binds deuteroporphyrin and 2,4-disulfonic acid deuteroporphyrin (dsDp) but not ethylenediamine-substituted protoporphyrin. Formation of the dsDp-hemopexin complex, produces a red shift in Soret maxima from 402 to 426 nm. Concomitant with this shift, the four-banded spectrum in the visible region changes to one with two absorption maxima at 554 and 590 nm. Fluorescence quenching data indicate the formation of an equimolar complex of hemopexin and dsDp with apparent dissociation constant of 1.8 × 10^?6m. The fluorescence emission maxima are at 623 nm for dsDp, at 603 nm for dsDp di-cation and at 590 nm for dsDp-hemopexin. The spectral changes following the interaction of dsDp with hemopexin may be interpreted to indicate that the porphyrin is in a less polar environment with an altered symmetry of the porphyrin nucleus. Since the dsDp-hemopexin spectrum resembles that of the di-cation we suggest that the pyrrole nitrogens of dsDp are protonated by or hydrogen bonded to two amino acids in the heme-binding site of hemopexin.     

The role of tissue interactions in the skeletogenic differentiation of avian neural crest cells

In the avian embryo, cranial neural crest (NC) cells migrate extensively throughout the head region and give rise to most of the cranial skeleton (Le Lievre, C. S. (1978). J. Embryol. Exp. Morphol.47, 17–37). To investigate the skeletogenic differentiation of these cells, NC explants from the mesencephalic level of st. 9+ embryos were grown in standard organ culture on Millipore filter substrates either in isolation or in combination with those tissues with which the cells normally associate during their in vivo migration and at their final tissue sites. The results demonstrate that interaction between premigratory NC and cranial ectoderm leads to chondrogenic differentiation of NC cells. Combination of premigratory NC with presumptive site tissues led to a pattern of NC cell differentiation normally expressed after in vivo migration: Combinations of NC with retinal pigmented epithelia gave cartilage, whereas NC with maxillary ectoderm formed cartilage and membrane bone. Both resulting skeletal tissues possessed their characteristic collagen types (II in cartilage and I in bone) as shown by indirect immunofluorescence using antibodies raised against specific types of collagen. It is concluded that avian cephalic NC cells require tissue interactions if chondrogenic and osteogenic differentiation is to ensue, but that migration per se is not an absolute prerequisite for these types of differentiation. The degree of specificity underlying such interactions is discussed.     

Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 RNA and specific 50 S subunit proteins

Pancreatic RNase partial digests of ³²P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed.     

Hormone mediated induction of acid phosphatase activity in the fat body of Calliphora erythrocephala prior to metamorphosis

At the end of the larval feeding stage of Calliphora erythrocephala, ecdysteroids are most likely to be responsible for the rapid increase in acid phosphatase activity in the fat body. This is demonstrated by the precocious induction of the enzyme by 20-hydroxyecdysone in ligatured feeding-stage larvae weighing 55–70 mg. The hormone does not influence normal protein accumulation: this is inhibited by the ligature and is not restored by injection of the hormone.     

Structure of magnesium paracrystals of α-tropomyosin

The molecular packing of magnesium paracrystals of α-tropomyosin has been examined by electron microscopy. Previous work (Caspar et al., 1969) had shown that these structures are composed of antiparallel arrays of molecules and we have now studied the relative positions of the molecules by matching the banding patterns of paracrystals positively stained with uranyl acetate to the sequence of the molecule. The overlap between the C-termini of the molecules in the unit cell is 175 ± 2 residues and the overlap of the N-termini lies in the range 107 to 122 residues. In the long overlap region (between C-termini), and probably also in the short overlap region, the molecular packing is such that the periodic zones of negative charge present in the sequence (Stewart & McLachlan, 1975) lie opposite one another. We propose that magnesium bridges between opposing negative charges contribute strongly to the stability of the structure. We confirm earlier work (Stewart, 1975b) on the absolute orientation of the molecules in the paracrystal: the troponin binding site on tropomyosin is approximately 130 Å from the C-terminus, and Cys190 is within 10 to 15 Å units of the C-C dyad.     

Investigations on the structural perturbations induced by the binding of mercuric chloride to L-glutamate dehydrogenase.

Three mercuric chloride binding sites are identified on l-glutamate dehydrogenase. In the presence of EDTA, the binding of two mercuric chloride molecules per subunit induces the dissociation of the polyhexamers into hexamers. The physical and catalytic properties of this modified hexamer are similar to those of the native enzyme. This induced dissociation of the enzyme is probably the result of an exclusive binding of the mercurial to the free hexamer, and the dissociation velocity does not appear to be rate limited by the binding reaction of the mercurials. The third mercuric chloride binding site is protected by both EDTA and l-glutamate. The binding of HgCl₂ to this site leads to the complete inactivation of the protein. There is no overlap between these modifications of l-glutamate dehydrogenase and the two previously described modifications of the enzyme by mercurial.