p62/p56 are cortical granule proteins that contribute to formation of the cortical granule envelope and play a role in mammalian preimplantation development

The purpose of this study was to identify specific cortical granule protein(s) that form the cortical granule envelope and examine their role(s) in fertilization and preimplantation development. The polyclonal antibody A-BL2 was used to show that the cortical granules of mice, rats, hamsters, cows, and pigs contain a pair of proteins designated p62/p56. These proteins are released from hamster cortical granules at fertilization and contribute to formation of the cortical granule envelope, an extracellular matrix present in the perivitelline space of fertilized mammalian oocytes. P62/p56 were present in the cortical granule envelope throughout preimplantation development and were found in blastomere cortices of 4-cell to blastocyst stage embryos. Hamster oocytes fertilized in vivo in the presence of A-BL2 were all monospermic, suggesting that p62/p56 do not function in blocking polyspermy. Likewise treatment of morula to blastocyst stage hamster embryos with A-BL2 had no effect on the implantation of blastocysts. However, cleavage divisions were inhibited in vivo in a dose-dependent manner when fertilized oocytes or 2-cell embryos were treated with A-BL2. Inhibition of cell division was more pronounced in 2-cell embryos than in fertilized oocytes. This study identifies p62/p56 as cortical granule proteins that contribute to the formation of the cortical granule envelope and further supports the idea that after their release at fertilization, p62/p56 function in regulating preimplantation development at the level of oocyte and blastomere cleavage.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

Kinetic intermediates in the formation of ordered complexes from cytochrome c fragments. Evidence that methionine ligation is a late event in the folding process

The reactions of ferric heme-containing fragments with apofragments to form ordered complexes resembling native horse heart cytochrome c have been studied under conditions which resolve the overall process into consecutive second order and first order kinetic steps. In the initial, second order step the two fragments combine to form an intermediate complex which exhibits tryptophan 59 fluorescence quenching similar to native cytochrome c, but which has not yet achieved the native ligation state of the heme iron. The existence of first order processes following the second order step is demonstrated by absorbance changes in the Soret region. the entire absorbance change at 695 nm, relating to ligation of the sulfur atom of methionine 80 to the heme iron, is also associated with these first order processes. Thus, ligation of methionine is a late event in this self ordering of the polypeptide chains. Since the conformational energy is assumed to distinctly decrease in this late process of folding (Parr, G.R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 2616-2623), it would follow that small spatial rearrangements of the polypeptide chains in the late stage of folding (as manifested by the ligation of methionine) are associated with a specific decrease in energy.     

Peptidylarginine deiminase (PAD) is a mouse cortical granule protein that plays a role in preimplantation embryonic development

Background  

While mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa cortical granule protein (p75) present in a subpopulation of mouse cortical granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.     

Evidence that a single GTP is used in the formation of 80 S initiation complexes.

Evidence is presented that the GTP initially bound in ternary complex (Met-tRNAf.GTP.eukaryotic initiation factor 2 (eIF-2)) is the same GTP that is hydrolyzed to allow joining of a 40 S preinitiation complex with 60 S subunits. This evidence was obtained by two quite dissimilar techniques. The first was a kinetic analysis of AUG-directed methionyl-puromycin synthesis using either eIF-2 of eIF-2A to direct the binding of Met-tRNAf to 40 S subunits. The second technique was the isolation of 40 S preinitiation complexes by Sepharose 6B chromatography and subsequent quantitation of GTP hydrolysis and methionyl-puromycin synthesis under conditions where 80 S complex formation is permitted.     

Evidence that pyrene excimer formation in membranes is not diffusion-controlled

Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant.     

Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.     

Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event.

A polyvalent antiserum which precipitates the native, folded, but not the denatured molecular forms of pig intestinal aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) was used to determine the kinetics of polypeptide folding of the two newly synthesized brush border enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a defective cotranslational high-mannose glycosylation, increased the half-time of polypeptide folding to about 12 min for aminopeptidase N as well as for sucrase-isomaltase. Short-pulse experiments suggested that fructose exerts its effect by slowing the rate of glycosylation, making this partially a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding.     

Evidence that autoimmunity in man is a Mendelian dominant trait.

Family studies of autoimmune diseases are consistent with multifactorial etiology. However, familial occurrence of the autoimmune trait as defined by the presence of autoimmune disease and/or high titer autoantibody supports the hypothesis that autoimmunity is inherited as an autosomal dominant trait. Based on genetic analysis of 18 autoimmune kindreds, the population frequency of this primary autoimmune gene is approximately .10 with penetrance estimates of 92% in females and 49% in males. The estimated high penetrance of the autoimmune gene in females suggests that the interacting genetic and/or environmental factors must be numerous or ubiquitous. Sex, age, and specific major histocompatibility complex (MHC) antigens are among the genetic and physiological factors known to influence autoimmunity. A genetic model is proposed that takes these factors into account. Inherent in the hypothesis of a primary autoimmune gene is that it is epistatic to other, secondary, genes that influence the autoimmune phenotype. The genetic model further postulates that the secondary genes, including those of the MHC, confer specificity to the phenotype. The effects of the secondary genes can be modulated by gonadal steroids and, over time, may be abrogated by environmental challenges, such as viral infections.     

Minireview. Evidence that dopamine is a neurotransmitter in peripheral tissues

In the CNS, dopamine (DA) is a recognized neurotransmitter as well as a precursor for norepinephrine (NE) and epinephrine (EPI). In contrast to the CNS, DA has been assumed to be only a precursor in peripheral tissues. There is now, however, considerable evidence to support the hypothesis that it may function as a neurotransmitter and/or cotransmitter in peripheral tissues in addition to being a precursor. In this minireview we summarize evidence supporting the view that DA plays a role of its own in peripheral neurotransmission.     

Evidence that polygalacturonase is a virulence determinant in Erwinia carotovora.

Polygalacturonase (PG) was purified from Erwinia carotovora EC. A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E. carotovora genomic library by an immunological screening method. A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices. In addition, the E. coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices. Our results suggest that PG plays an important role in soft-rot disease.     

Evidence that the Mg-ATPase in the cortical layer of sea urchin egg is dynein

Indirect immunofluorescence staining of cleaving sea urchin eggs with an antiserum against a tryptic fragment of dynein 1 (fragment 1A) from sea urchin sperm flagella suggested the presence of dynein in the cortex as well as in the mitotic apparatus. In the present study, we found that the Mg²⁺-ATPase activity of the isolated cortices from sea urchin eggs, which exhibited similar characteristics to those of flagellar dynein, was inhibited by 60–80% with the anti-fragment 1A serum. Faintly stained bands corresponding to the A-band (dynein 1) and the B-band of the sperm flagella was detected on sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the isolated cortices. Furthermore, the SDS-gel electrophoresis revealed the presence of a polypeptide band corresponding to dynein 1 in the antigen-antibody complex precipitated from the KCl-extract of the cortices with the antiserum.     

Mitochondrial gene expression in mammalian striated muscle. Evidence that variation in gene dosage is the major regulatory event

The oxidative capacity of mammalian striated muscles can vary markedly over a nearly 10-fold range, reflecting major differences in the expression of genes that encode enzymes of oxidative metabolism, including genes located exclusively within mitochondrial DNA. To clarify the regulatory events that govern expression of mitochondrial genes in striated muscle, nucleic acid hybridization procedures employing cloned segments of mitochondrial DNA as probes were utilized to determine the concentrations of mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA (a mitochondrial gene product) in rabbit striated muscles of markedly different oxidative capacities. When cardiac muscle and Type I (red, oxidative) skeletal muscle were compared to Type II (white, glycolytic) skeletal muscle, mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA, each increased in direct proportion to increases in oxidative capacity. Furthermore, when the phenotypic characteristics of Type II skeletal muscle were altered by electrical stimulation in vivo, mitochondrial DNA, mitochondrial rRNA, and cytochrome b mRNA also increased proportionately with increases in oxidative capacity. These results indicate that the expression of mitochondrial genes in mammalian striated muscle is proportionate to their copy number, and support the hypothesis that amplification of the mitochondrial genome relative to chromosomal DNA is an important feature underlying enhanced expression of mitochondrial genes in highly oxidative tissues.     

Ym1 is a neutrophil granule protein that crystallizes in p47phox-deficient mice.

Crystals were discovered within the aged lung and at sites of chronic inflammation in a mouse model of chronic granulomatous disease. Following re-crystallization at neutral pH, the crystals were identified as the chitinase-like protein Ym1, expressed in organs of the lymphoreticular system, the lung, and distal stomach. Ym1 was shown to be a neutrophil granule protein and to have weak beta-N-acetylglucosaminidase activity, indicating that it might contribute to the digestion of glycosaminoglycans. Crystal formation is likely to be a function of excess neutrophil turnover at sites of inflammation in the chronic granulomatous disease mouse. Failure to remove subcutaneous Ym1 crystals injected into knockout mice indicates that a failure of digestion may also contribute to crystallization.     

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.