Stathmin expression in the Placenta and Embryonic Brain.ABSTRACT. Stathmin is a member of a family of proteins believed to play important roles in neural development. In the current study, expression of stathmin forms in rat brain was examined from day 15 of gestation through the neonatal period and into adulthood. Placental placental pertaining to or emanating from placenta. placental barrier the placental separation of maternal and fetal blood which varies in its structure and permeability between the species. stathmin also was analyzed. Unphosphorylated and phosphorylated forms of stathmin in the brain are highest in the late fetal and early neonatal period. Placenta expresses some larger stathmin-like proteins, but none of the 19 kilodalton forms of stathmin. The results show that stathmin is greatly elevated during a period of rapid brain growth and development, suggesting that stathmin plays a regulatory role in development. The functional significance of the unique pattern of stathmin-like proteins expressed by the placenta is unclear at this time. Keywords: Stathmin, rat, brain, development, placenta Stathmin is a cytosolic phosphoprotein phosphoprotein /phos·pho·pro·tein/ (-pro´ten) a conjugated protein in which phosphoric acid is esterified with a hydroxy amino acid. phos·pho·pro·tein n. believed to be involved in intracellular signaling (Sobel 1991). Though found in almost all tissues, stathmin is expressed most highly in the nervous system (Amat et al. 1991; Doye et al. 1992; Koppel et al. 1990; Maucuer et al. 1993; Schubart 1988; Sugiura & Mora MORA, In civil law. This term, in mora, is used to denote that a party to a contract, who is obliged to do anything, has neglected to perform it, and is in default. Story on Bailm. Sec. 123, 259; Jones on Bailm. 70; Poth. Pret a Usage, c. 2, Sec. 2, art. 2, n. 1995). It is a member of a family of proteins that in mammals includes SCG SCG Serbia and Montenegro SCG Srbija I Crna Gora (Servian: Serbia and Montenegro) SCG Sydney Cricket Ground SCG Service Canadien des Glaces (Canadian Ice Service) SCG superior cervical ganglion 10 (Anderson & Axel 1985; Stein et al. 1988), SCG10-like protein (SCLIP) (Ozon et al. 1998) and RB3 (Ozon et al. 1997). These latter family members are almost exclusively expressed in neural tissue (Ozon et al. 1997; Ozon et al. 1998). The expression of stathmin and family members in neural tissue supports a regulatory role for these proteins in development of the nervous system. Though it is likely that members of the stathmin family have overlapping functions, they probably play distinct regulatory roles in neural development, given their differences in distribution within the nervous system (Ozon et al. 1997; Ozon et al. 1998), developmental expression (Amat et al. 1991; Anderson & Axel 1985; Doye et al. 1992; Koppel et al. 1990; Maucuer et al. 1993; Ozon et al. 1997; Ozon et al. 1998; Schubart 1988; Sugiura & Mora 1995) and cytosolic vs. membrane localization Customizing software and documentation for a particular country. It includes the translation of menus and messages into the native spoken language as well as changes in the user interface to accommodate different alphabets and culture. See internationalization and l10n. (Anderson & Axel 1985; Di Paolo et al. 1997; Ozon et al. 1997; Ozon et al. 1998; Sobel 1991). Several reports (Amat et al. 1991; Doye et al. 1992; Koppel et al. 1990; Maucuer et al. 1993; Schubart 1988; Sugiura & Mora 1995) have suggested that stathmin expression in the brain is high in the late embryonic and early neonatal period, and then declines into adulthood. Ozon et al. (1998) have demonstrated that expression of stathmin mRNA increases in the rat brain from day 16 of gestation to a peak in the early neonatal period and then decreases toward adulthood. The study also showed that expression of SCG10 mRNA followed a similar developmental pattern in the brain. In contrast, SCLIP and RB3 mRNAs did not decline after the neonatal period. In the current study, we have examined the forms of stathmin expressed in the brains of embryonic, neonatal and pregnant-adult rats and in rat placenta. The data confirm that stathmin is highest in the late embryonic and early neonatal period. Furthermore, the data suggest that the placenta expresses larger stathmin-like proteins, but few if any 19 kilodalton (kDa) forms of stathmin. METHODS Animals/tissues.--Tissues were obtained from Sprague-Dawley rats. Adult animals weighed between 180-300 g. Brains from a minimum of five separate animals were pooled for studies of fetal and neonatal stathmin. The 10-day preparation was prepared from multiple placenta-fetus samples taken from two different pregnant rats. All preparations from adult animals involved tissues obtained from at least two separate animals. Anti-stathmin antibody.--The anti-stathmin antiserum antiserum /an·ti·se·rum/ (an´ti-se?rum) a serum containing antibody(ies), obtained from an animal immunized either by injection of antigen or by infection with microorganisms containing antigen. was generated in rabbits against amino-acid residues 32-44 (VPEFPL SPPKKKD) of rat stathmin (Flurkey et al. 1993; Meyer et. al. 1992). This sequence is highly conserved among mammalian stathmins, but it is less conserved among the stathmin family members SCG10 (EAPRTLASPKKKD), SLIP (PESPVLSSPPKRKD) and RB3 (VPEFNASLPRRRD). The carboxy-terminal five amino acids of the fragment used to generate the antibody are shared by stathmin and SCG10, so some cross-reactivity is possible. In the immunostaining procedure, the antibody was used at a concentration of 1:5000. Stathmin extraction.--Whole rat brains were suspended in 3 volumes of an ice-cold extraction buffer consisting of 100 mM Tris-HCI, 1 mM phenylmethylsulfonyl fluoride, 10 [micro]/mL leupeptin and 0.02% sodium azide sodium azide NaN3 Microbiology A toxic salt added–concentration, 0.01%, to a transport medium of lab specimens–eg, urine for culturing bacteria, which prevents oxidative phosphorylation and bacterial overgrowth at pH 7.0. The suspension was sonicated using three 5-sec bursts at a setting of 2.3 on a Branson Sonifier (Branson Ultrasonic Corp., Danbury, Connecticut). The suspension was centrifuged at 800 x g at 40[degree]C for 4 min, and the supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material. supernatant the liquid lying above a layer of precipitated insoluble material. fraction was transferred to a 1.5 mL microfuge tube. Sodium chloride sodium chloride, NaCl, common salt. Properties Sodium chloride is readily soluble in water and insoluble or only slightly soluble in most other liquids. It forms small, transparent, colorless to white cubic crystals. (1 M, in 100 mM Tris-HCI, pH 7.0) was added to a final concentration of 100 mM and the solution was heated to 100[degree]C for 10 min. The tubes were placed on ice for 5 min and centrifuged at 12,000 X g for 5 min, and the stathmin-enriched supernatant fraction was transferred to a clean 1.5 mL microfuge tube. Protein was measured using a colorimetric col·or·im·e·ter n. 1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards. 2. assay (BCA BCA Business Case Analysis BCA Building Code of Australia BCA Boeing Commercial Airplanes BCA Board of Contract Appeals BCA Boston Center for the Arts BCA Billiard Congress of America BCA Bureau of Criminal Apprehension BCA Breast Cancer Action Protein Assay, Pierce, Rockford, Illinois). Electrophoresis.--Samples were analyzed by sodium dodecyl sulfate-polyacrylatnide gel electrophoresis (SDS-PAGE SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. , Laemmli, 1970) on 12% polyacrylamide gels (Bio-Rad Laboratories). Samples (35-70 [micro]g protein) were combined with an equal volume of 2X SDS 1. (company) SDS - Scientific Data Systems. 2. (tool) SDS - Schema Definition Set. sample buffer (0.125 M Tris-HCI, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. , 0.05% bromophenol blue, pH 6.8) and heated to 100[degree]C for 10 min. Electrophoresis was run at a constant current of 55 mA for approximately 4 h. Proteins in the gels were electrophoretically transferred (60 V, 1 h) to nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. paper (0.22 [micro]m) using a Trans-Blot Cell (Bio-Rad Laboratories) (Towbin et al. 1979). Samples frozen at -70[degree] C were reanalyzed within 2-3 weeks to confirm results. Immunostaining.--Open sites on the nitrocellulose paper were blocked by incubating the paper in binding buffer (100 mM] Tris-HCI, 0.15 M NaC1, 0.1% BSA 1. BSA - Business Software Alliance. 2. BSA - Bidouilleurs Sans Argent. , pH 8.0) containing 5% non-fat dry milk. After three 10-min washes in binding buffer, the nitrocellulose was incubated for 22 h in binding buffer containing the anti-stathmin antiserum (1: 5000) (Flurkey et al. 1993; Meyer et. al. 1992). Unbound unbound said of electrolytes, e.g. iron and calcium, and other substances which are circulating in the bloodstream and are not bound to plasma proteins so that they are available immediately for metabolic processes. See also calcium, iron. antibody was removed by three 10-min washes in binding buffer, and the nitrocellulose was incubated for 1 h with a goat anti-rabbit gamma globulin gamma globulin, a group of globulin proteins in human blood plasma, including most antibodies. These antibody substances are produced as a protective reaction of the body's immune system to the invasion of disease-producing organisms (see immunity). coupled to alkaline phosphatase alkaline phosphatase /al·ka·line phos·pha·tase/ (ALP) (fos´fah-tas) an enzyme that catalyzes the cleavage of orthophosphate from orthophosphoric monoesters under alkaline conditions. (Sigma Chemical Company, St. Louis, Missouri). Unbound antibody was removed by three 10-min washes in binding buffer, and the alkaline phosphatase was detected using the BCIP/NBT (Pierce Chemical Co., Rockford Illinois). Density analysis.--Immunostained bands were scanned using an Arcus II scanner and were quantified using the ONE-Dscan program (Scanalytics). Relative stathmin units were calculated by comparing the optical densities of the samples to those of aliquots (5 [micro]l, 7.5 [micro]l and 15 [micro]l of purified stathmin. RESULTS The results show that brain stathmin levels increase from late gestation to the early neonatal period and then decline in older animals (Fig. 1). High levels were present in brains obtained from rats at 2, 5 and 10 days after parturition parturition or birth or childbirth or labour or delivery Process of bringing forth a child from the uterus, ending pregnancy. It has three stages. . Most of the stathmin migrated with a molecular weight of approximately 19,000 (Fig. 1), consistent with unphosphorylated and less-phosphorylated forms of stathmin. However, higher molecular-weight forms (23-24 kDa), consistent with highly phosphorylated stathmin, were observed in brains taken from all fetal and neonatal rats. Lower stathmin levels were found in extracts prepared using brains obtained from nulliparous adult rats and higher molecular-weight forms were not detected (Fig. 1). No 19 kDa forms of stathmin were detected in placenta/fetal tissue from a 10-day pregnant rat. A single 23 kDa protein was detected using the antibody. Fetal and placental tissues could not be reliably separated at this stage of gestation, so the preparation contained elements of both. However, the high molecular weight protein is likely of placental origin, because placenta accounted for most of the tissue at this stage. Stathmin was present at a high concentration in brains obtained from fetal rats at 20-days gestation, the expected day of parturition (Fig. 2). Immunostaining of proteins from the 20-day fetus was considerably greater than that of proteins from the 10-day placental/fetal preparation, a preparation that also is presented in Fig. 1 for comparison. Based on its staining relative to the 10-day placental/fetal preparation, the 20-day fetal brain (Fig. 2) contained stathmin at levels comparable to those found in the 2-day neonatal animal (Fig. 1). Higher molecular-weight forms of stathmin in the 20-day fetal brain also were similar to those detected in the brain of the 2 day neonate neonate /neo·nate/ (ne´o-nat) newborn infant. ne·o·nate n. A neonatal infant. neonate a newborn animal. . Brain from a 20-day pregnant rat showed almost equal amounts of immunostaining at 19 and 23 kDa (Fig. 2). The appearance of a 23 kDa band is interesting, because no comparable protein was detected in the adult nulliparous animals. The 23 kDa protein appeared to correspond with one of the higher molecular-weight proteins immunostained in extracts from the 20-day fetal brain, 20-day placenta and the 10-day placenta/fetus. The extract from the 20-day placenta did not show evidence of 19 kDa forms of stathmin, in agreement with results obtained with the 10-day placenta/fetus. A 30 kDa protein also was identified in the 20-day placenta. The immunostained bands in Fig. 2 were analyzed using the ONE-Dscan program (Scanalytics) to obtain an estimate of integrated optical density. The optical densities were then compared to those of different amounts (5 [micro]l 7.5 [micro]l and 15 [micro]l) of a purified stathmin (Fig. 2), so that staining could be expressed as arbitrary stathmin units. As shown in Table 1, brain tissue from the 20-day fetus contained approximately 10 times as much stathmin-like substance, most of which migrated as a 19 kDa protein(s). DISCUSSION The data show that expression of stathmin protein in the rat brain is highest in the late fetal and early neonatal period and then declines into adulthood. In fact, brain stathmin peaks in the period encompassed by day 20 of gestation to day 2 after parturition. This developmental pattern closely parallels that reported (Ozon et al. 1998) for expression of stathmin mRNA in rat brain. The results also are consistent with data from earlier studies (Amat et al. 1991; Doye et al. 1992; Koppel et al. 1990; Maucuer et al. 1993; Schubart 1988; Sugiura & Mora 1995) showing higher levels of stathmin in brains from neonatal animals. Larger (> 19 kDa) stathmin-like proteins were identified in nearly all brain extracts. Most of these forms probably represent phosphorylated stathmin, because it has been demonstrated previously (Beretta be·ret·ta or ber·ret·ta n. Variants of biretta. et al. 1993; Cardinaux et al. 1997; Chneiweiss et al. 1989; Chneiweiss et al. 1992; Doye et al. 1992) that brain contains phosphorylated forms of stathmin that migrate with apparent molecular weights of 21,000-25,000. Though larger forms were not detected in a nulliparous adult female rat, the phosphorylated forms likely were present, but at low levels. A 30 kDa form(s) identified in placenta from a 20-day pregnant rat is larger than would be expected for phosphorylated stathmin. This form might represent one of the stathmin-related proteins such as SCG10, SLIP or RB3. The most likely candidate would be SCG10 since this protein shares some homology with stathmin in the region of the molecule used to generate the anti-stathmin antibody (please see Methods), and SCG10 is present at a relatively high concentrati on in brain. However, SCG10 usually migrates with an apparent molecular weight less than 30,000 (Ozon et al. 1997). Using an anti-RB3 antiserum, Ozon et. al. (1997) reported unidentified "X" proteins that migrate with an apparent molecular weight of 30,000. Hence, the 30 kDa protein could be a yet unidentified stathmin-like protein, perhaps related to RB3. No 19 kDa stathmin forms were detected in placenta. The smallest form detected in placental tissue obtained at 10 and 20 days of gestation was approximately 23 kDa. Interestingly, a similar-sized protein was prominent in the brain obtained from a 20-day pregnant animal. The dearth of 19 kDa forms of stathmin in placenta may suggest that placental enzymes actively phosphorylate phos·pho·ryl·ate tr.v. phos·pho·ryl·at·ed, phos·pho·ryl·at·ing, phos·pho·ryl·ates To add a phosphate group to (an organic molecule). phos stathmin. Alternatively, the protein immunostained at 23 kDa may represent SCG10 or other stathmin-related protein. In any case, it is remarkable that a tissue undergoing growth and development contains so little unphosphorylated stathmin. In summary, the results show that expression of stathmin in brain is highest close to the time of parturition. The data also show that the pattern of "stathmin" forms expressed by placental tissue differs from that in brain. LITERATURE CITED Amat, J.A., K.L. Fields & U.K. Schubart. 1991. Distribution of phosphoprotein p19 in rat brain during ontogeny ontogeny: see biogenetic law. Ontogeny The developmental history of an organism from its origin to maturity. It starts with fertilization and ends with the attainment of an adult state, usually expressed in terms of both maximal body : Stage-specific expression in neurons and glia. Developmental Brain Research 60:205-218. Anderson, D.J. & R. Axel. 1985. Molecular probes for the development and plasticity of neural crest Neural crest A strip of ectodermal material in the early vertebrate embryo inserted between the prospective neural plate and epidermis. After closure of the neural tube the crest cells migrate into the body and give rise to parts of the neural system: the main derivatives. Cell 42:649-662. Beretta L., T. Dobransky & A. Sobel. 1993. Multiple phosphorylation phosphorylation, chemical process in which a phosphate group is added to an organic molecule. In living cells phosphorylation is associated with respiration, which takes place in the cell's mitochondria, and photosynthesis, which takes place in the chloroplasts. of stathmin. Identification of four sites phosphorylated in intact cells and in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. by cyclic AMP-dependent protein kinase protein kinase /pro·tein ki·nase/ (pro´ten ki´nas) an enzyme that catalyzes the phosphorylation of serine, threonine, or tyrosine groups in enzymes or other proteins, using ATP as a phosphate donor. and p34cdc2. Journal of Biological Chemistry The Journal of Biological Chemistry (often abbreviated JBC) is a scientific journal founded in 1905 and published since 1925 by the American Society for Biochemistry and Molecular Biology. 268:20076-20084. Cardinaux, J.-R., P.J. Magistretti & J.-L. Martin. 1997. Brain-derived neurotrophic factor Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor found in the brain and the periphery. It is a protein that acts on certain neurons of the central nervous system and the peripheral nervous system that helps to support the survival of existing neurons and encourage stimulates phosphorylation of stathmin in cortical neurons. Molecular Brain Research 51:220-228. Chneiweiss H., L. Beretta, J. Cordier, M.C. Boutterin, J. Glowinski & A. Sobel. 1989. Stathmin is a major phosphoprotein and cyclic AMP-dependent protein kinase substrate in mouse brain neurons but not in astrocytes astrocytes (as´trōsī´ts), n a large, star-shaped cell found in certain tissues of the nervous system. A mass of astrocytes is called astroglia. See also astrocytoma. in culture: Regulation during ontogenesis ontogenesis /on·to·gen·e·sis/ (on?to-jen´e-sis) ontogeny. on·to·gen·e·sis n. See ontogeny. . Journal of Neurochemistry neurochemistry /neu·ro·chem·is·try/ (-kem´is-tre) the branch of neurology dealing with the chemistry of the nervous system. neu·ro·chem·is·try n. 53:856-863. Chneiweiss H., J. Cordier & A. Sobel. 1992. Stathmin phosphorylation is regulated in striatal neurons by vasoactive intestinal peptide Vasoactive intestinal peptide (VIP, also polypeptide[1]) is a peptide hormone containing 28 amino acid residues and is produced in many areas of the human body including the gut, pancreas and suprachiasmatic nuclei of the hypothalamus in the brain. and monoamines via multiple intracellular pathways. Journal of Neurochemistry 58:282-289. Di Paolo, G., R. Lutjens, V. Pellier, S.A. Stimpson, M.A. Beuchat, M. Catsicas & G. Grenningloh. 1997. Targeting of SCG10 to the area of the Golgi complex Golgi complex n. A complex of parallel, flattened saccules, vesicles, and vacuoles that lies adjacent to the nucleus of a cell and is concerned with the formation of secretions within the cell. Also called Golgi apparatus. is mediated by its [NH.sub.2]-terminal region. Journal of Biological Chemistry 272:5175-5182. Doye, V., S. Le Gouvello, T. Dobransky, H. Chneiweiss, L. Beretta & A. Sobel. 1992. Expression of transfected stathmin cDNA reveals novel phosphorylated forms associated with developmental and functional cell regulation. Biochemical Journal 287:549-554. Doye, V., O. Kellerman, M.-H. Buc-Caron & A. Sobel. 1992. High expression of stathmin in multipotential teratocarcinoma teratocarcinoma /ter·a·to·car·ci·no·ma/ (-kahr?si-no´mah) a malignant neoplasm consisting of elements of teratoma with those of embryonal carcinoma or choriocarcinoma, or both; occurring most often in the testis. and normal embryonic cells versus their early differentiated derivatives. Differentiation 50:89-96. Flurkey, W.H., D.A. Prentice, M.T. Fox & J.P. Hughes. 1993. Stathmin in mung bean mung bean n. 1. An Asian plant (Vigna radiata) in the pea family, widely cultivated for its edible seeds and pods. It is the chief source of bean sprouts. 2. The seeds or pods of this plant. leaves and rat brain. Biochemical and Biophysical Research Communications 196:589-595. Koppel, J., M.-C. Boutterin, V. Doye, H. PeyroSaint-Paul & A. Sobel. 1990. Developmental tissue expression and phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. conservation of stathmin, a phosphoprotein associated with cell regulations. Journal of Biological Chemistry 265:3703-3707. Laemmli, U.K. 1970. Cleavage of structural proteins during assembly of bacteriophage T4. Nature 227:680-685. Maucuer, A., J. Moreau, M. Mechali & A. Sobel. 1993. Stathmin gene family: Phylogenetic conservation and developmental regulation in Xenopus. Journal of Biological Chemistry 268: 16420-16429. Meyer, N., D.A. Prentice, M.T. Fox & J.P. Hughes. 1992. Prolactin-induced proliferation of the Nb2 T-lymphoma is associated with protein kinase-C-independent phosphorylation of stathmin. Endocrinology 131:1977-1984. Ozon, S., T. Byk & A. Sobel. 1998. SCLIP: A novel SCG10-like protein of the stathmin family expressed in the nervous system. Journal of Neurochemistry 70:2386-2396. Ozon, S., A. Maucuer & A. Sobel. 1997. The stathmin family: Molecular and biological characterization of novel mammalian proteins expressed in the nervous system. European Journal of Biochemistry 248:794-806. Schubart, U.K. 1988. Expression of phosphoprotein p19 in brain, testis testis (tĕs`tĭs) or testicle (tĕs`tĭkəl), one of a pair of glands that produce the male reproductive cells, or sperm. , and neuroendocrine tumor neuroendocrine tumor Neuropathology A neoplasm with a characteristic morphology, often composed of clusters and trabecular sheets of round 'blue cells', granular chromatin, and an attenuated rim of poorly demarcated cytoplasm Examples Carcinoids, small–'oat' cells: Developmental regulation in rat brain. Journal of Biological Chemistry 263:12156-12160. Sobel, A. 1991. Stathmin: A relay phosphoprotein for multiple signal transduction? Trends in Biochemical Science 16:301-305. Stein, R., N. Mori, K. Matthews, L.C. Lo & D.J. Anderson. 1988. The NGF-inducible SCG10 mRNA encodes a novel membrane-bound protein present in growth cones and abundant in developing neurons. Neuron 1:463-476. Sugiura, Y. & N. Mora. 1995. SCG10 expresses growth-associated manner in developing rat brain, but shows a different pattern to p19/stathmin or GAP-43. Developmental Brain Research 90:73-91. Towbin, H., T. Staehelin & J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Science USA 76:4350-4354.
Table 1
Stathmin in brain and placenta.
Stathmin
Stathmin amount
forms (reltive
Tissue (MW, kDa) units) (1)
Brain
20 Day Pregnant 19 1.8
23 1.4
19 17.6
23 1.4
24 0.9
20 Day Fetus 27 0.0 (2)
Placenta
23 0.8
20 Day 30 1.3
10 Day 23 0.8
(1)Units expressed relative to immunostaining of a purified stathmin
(see Methods).
(2)Band could not be accurately quantified by the ONE-Dscan program
(Scanalytics).
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