Synthesis of tubulin and actin during the preimplantation development of the mouse

The relative rates of synthesis of actin and tubulin during mouse preimplantation development have been investigated utilizing O'Farrell's two-dimensional polyacrylamide gel system and internal protein markers. During mouse preimplantation development, rates of protein synthesis remain low and are little changed until the 8-cell stage when a rapid increase is evident. From the 8-cell stage on, a much higher rate of synthesis is maintained. The rate of synthesis of actin remains also at a steady low level in the unfertilized and fertilized ovum. However, by the 8-cell stage actin synthesis has increased 10-fold. Our measurements include the blastocyst, at that point in development actin synthetic rates are almost 90-fold higher than in the unfertilized ovum. While this precipitous increase is proceeding, incorporation of [³H]leucine into total protein increases only 7-fold. Synthesis of actin in the blastocyst represents 5.7% of total protein synthesis. The rate of tubulin synthesis, unlike actin, more closely parallels the increments in total protein synthetic rates. At the blastocyst stage it has increased 14-fold and its synthesis represents almost 2% of total protein synthesis. These results are discussed with reference to some of the physiological changes taking place during preimplantation development.     

Reduction of anionic sites on the rabbit uterine surface during the preimplantation period

Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5–7 of pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.     

Specific binding of sulfated proteoglycans to concanavalin A - Sepharose 4B.

More than 90 % of [³⁵S]proteoglycans isolated from the secretions of human skin fibroblasts bind to Concanavalin A-Sepharose 4B (Con A-Sepharose) in the presence of 1 M NaCl. Above pH 5.0 1 M concentrations of methyl-α-D-mannoside and other haptenic inhibitors for Con A-sugar interaction prevent binding of [³⁵S]proteoglycans, whereas equimolar concentrations of non-haptenic carbohydrates do not effect binding. Below pH 5.0 [³⁵S]proteoglycans bind to Con A-Sepharose in the presence of both methyl-α-D-mannoside and galactose. About 60 % of the proteoglycans bound at pH 4.0 are eluted at pH 7.5 in the presence of 1 M methyl-α-D-mannoside. [³⁵S] Glycosaminoglycans prepared from [³⁵S] proteoglycans do not bind to Con A-Sepharose in the presence of 1 M NaCl.These results indicate a [³⁵S]proteoglycan-Con A interaction via the protein core of the proteoglycan and the sugar binding sites of Con A.     

A Co(III) derivative of concanavalin A

Co(III) has been stoichiometrically incorporated into jack bean concanavalin A. The Co(III) protein still possesses a binding site for an additional divalent transition metal ion which together with Ca(II) can induce the sugar binding ability. No H₂O₂ oxidation of Co(II) occurs with demetallized concanavalin A activated with Ca(II) and Co(II) unless Co(II) is present in a stoichiometric excess. Evidence is presented to indicate that kinetically stable Co(III) is bound to a completely different location than the thermodynamically stable Co(II) protein site.     

Fertilization-induced changes in concanavalin A binding to mouse eggs

The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml^?1) the fertilized egg shows a higher affinity for [³H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 10⁸ ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.     

Cytochrome changes in control and concanavalin A-stimulated lymphocytes

α-Aminoisobutyrate (AIB) serves as a transportable, nonmetabolizable alanine analog in the purple sulfur bacterium Chromatium vinosum. AIB transport in C. vinosum appears to be catalyzed by an electrogenic Na⁺-alanine (AIB) symport without any direct participation of ATP-driven or H⁺-symport systems. In addition to Na⁺ being cotransported with AIB via the symport, a transmembrane Na⁺ gradient appears to increase the affinity of the symport of AIB. It appears that these two effects of Na⁺ involve different Na⁺-binding sites.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.     

The biochemical and ultrastructural demonstration of collagen during early heart development

A correlative ultrastructural and biochemical study was made of cardiac collagen in the chick embryo, spanning stages 9- to 11 (6 to 13 somites). Analysis (carboxymethyl cellulose chromatography, SDS acrylamide gel electrophoresis and levels of proline hydroxylation) of collagen synthesized in situ permitted classification of this collagen as type I-like (α1:α2 = 2:1). Correlative electron microscopy of hearts fixed in situ showed the appearance of striated collagen fibrils in the cardiac jelly, thus complementing the biochemical findings. Although the electron microscope showed the presence of developing basal laminae and laminalike material, synthesis of the type of collagen reported as unique to basal laminae was not detected, and we propose that these basal laminae may lack type IV collagen. Embryonic stages 9- to 11 are the earliest stages in which collagen synthesis has been demonstrated, and this is the first report of the occurrence of collagen in cardiac jelly of early hearts.     

The effect of short chain fatty acids on transmural electrical potential across rat small intestine in vivo

Short chain fatty acids suddenly produce a phasic increase in transmural electrical potential difference (PD) when placed in the lumen of rat small intestine in vivo. With concentrations of propionate ranging fro 50μM to 1000 μM the amplitude of the response in jejunum is about 5.5 mV. The concentration giving half this effect is about 20 μM. With 10 mM propionate the duration of the response is 3–5 min; after this, PD again equals the control value and the gut is refractory to further additions. Removing propionate from the mucosal surface produces no change in PD, but does restore responsiveness to subsequent exposure to short chain fatty acids.This effect is indpendent of a variety of other alterations in PD such as those caused by sugars, amino acids, bile salts, theophylline, prostaglandins, and ATP. Mechanism and significance of this surprisingly sensitive response remain obscure.     

The absence of cell death during development of free digits in amphibians

Massive cellular death occurs in the interdigital regions of developing limbs of free-digited birds and mammals. This mesodermal degeneration occurs at the same time that digits become free. The present study of digit formation in amphibians, using vital staining and histological and autoradiographic techniques, demonstrates the absence of zones of interdigital degeneration during the formation of free digits. Furthermore, no other areas of predictable cell death occur during amphibian limb development, a situation quite unlike the case for avian limb development where predictable zones of degeneration occur in the mesoderm along the pre- and postaxial borders of the developing wing and leg. Thus, zones of cell death are not a part of amphibian limb morphogenesis. Analysis of the labeling index of the developing free-digited forelimb of Xenopus laevis reveals that during stage 52 the interdigital and digital labeling indexes are the same. The change in the ratio of interdigital labeling index to the digital labeling index in the forelimb suggests that during subsequent development the interdigital labeling index decreases while the digital labeling index is maintained. In comparison, the same analysis indicates that the interdigital labeling index of the webbed hindlimb increases when compared to the digital labeling index, which stays the same from early to late stages. It is proposed that free digits develop in Xenopus laevis forelimb as a result of a decrease in the proliferation rate of the interdigital region as compared to the digital region, which remains unchanged. Conversely, webbed digits develop in the hindlimb as a result of an interdigital rate at least equal to the digital rate.     

Conformational changes following Mn(II) binding to demetalized concanavalin A

A temperature-dependent conformational change occurs following the binding of only one Mn(II) to a concanavalin A monomer. This change is independent of Ca(II) near pH 7 and is characterized by an activation energy of 22.3 kcal mol^?1, a value similar to that attributed to a cis-trans peptide isomerization. Two conformations have been detected in magnetic resonance experiments on solvent water protons where spin lattice relaxation times are influenced by bound Mn(II). Both conformations possess saccharide binding activity and Ca(II) stoichiometrically enhances the rate of conversion to the final, more stable conformation.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF₇, ZR₇₅ and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of ³⁵S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF₇ cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.     

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.