Na+-dependent amino acid transport in preimplantation mouse embryos. II. Metabolic inhibitors and nature of the cation requirement.

Experiments were conducted in order to determine the energy source and nature of the cation dependency of [³H]methionine transport in preimplantation mouse embryos. The energy source of methionine transport was studied at the late four-cell and early blastocyst stages. The embryos, raised in vitro, were incubated for 1 hr in inhibitor(s) of energy metabolism and then transferred for 1 hr to medium that contained inhibitor(s) and ³H-methionine. These inhibitor studies suggest that respiration and glycolysis are needed to maintain uptake of methionine in early blastocysts. Late four-cell embryos seem to utilize respiration alone for transport.The cation dependency of methionine transport was studied at the late morula and early blastocyst stages. The kinetics of methionine uptake by early blastocysts in Na⁺-depleted media indicate a competitive type of inhibition. The uptake of methionine by early blastocysts is relatively resistant to ouabain and unaffected by K⁺-free medium. In contrast, methionine uptake by late morula-stage embryos is markedly inhibited by ouabain and K⁺-free medium in 1 hr. These results suggest that 1) Na⁺ serves to increase the affinity of methionine for the carrier in early blastocysts, 2) the cation gradients do not supply a major fraction of the energy required for methionine transport, and/or the gradients are difficult to perturb once the blastocyst has formed, and 3) putative Na⁺ pumps may be localized on the blastocoelic surface of the blastocysts.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

Synthesis of RNA containing polyadenylic acid sequences in preimplantation rabbit embryos

Messenger RNA synthesis has been estimated by assaying polyadenylic acid (poly A)-rich sequences in heterogeneous RNA from preimplantation rabbit embryos. Poly A containing RNAs are synthesized at least as early as the 16-cell stage and continue to be made through blastocyst formation and maturation. Sixty to 78% of the heterogeneous polysomal RNA in blastocysts contain poly A sequences. The portion of the heterogeneous RNA containing poly A sequences does not appear to change markedly between cleavage and blastocyst stages of development. Poly Arich sequences are greater than 4 S and consist of at least 84% adenine residues. RNA molecules ranging from 6 S to greater than 28 S contain poly A sequences.     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

The amino acid composition of secreted mouse prolactin,growth hormone,and hamster prolactin: The presence of one tryptophan in mouse prolactin

The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R_f's of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.     

Role of proton dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cells

The pH profile for the uptake of l-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na⁺-dependent uptake by System A, and an increasing uptake by Na⁺-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na⁺-dependent component. (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicuous exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another.A small Na⁺-dependent component of uptake retained by l-glutamic acid but not by d-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports l-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic acid or of the norbornane amino acid.     

The amino acid sequence of carbonic anhydrase I from the rhesus macaque

The complete amino acid sequence of carbonic anhydrase I (CA I) isolated from the red cells of the rhesus macaque ($\text{Macaca}$$\text{mulatta}$) is presented. This sequence was obtained by aligning peptides derived from various fragmentation procedures with the fully characterized sequence of human CA I. When the peptides of rhesus CA I were ordered in this manner, 13 of the 260 residues were found to differ from the human CA I sequence. The known markedly higher specific esterase activity of rhesus CA I compared to human CA I could not be correlated with any changes in residues postulated to be within 10 Å of the single zinc ion at the active site.     

Studies on the heat-precipitated phosphoenzyme complex of eel electric organ (Na + K).ATPase.

When the hydrolytic reaction between eel electric organ (Na + K) · ATPase and [γ-³²P]ATP is terminated at neutral pH by heat precipitation, a phosphoenzyme complex is formed which reaches maximal levels in the simultaneous presence of Mg, Na, and K. After formation of a steady-state level of phosphoenzyme in the presence of Mg and Na, a pulse of K increases the level of the heat-precipitated phosphoenzyme (while decreasing the level of the acid-precipitated phosphoenzyme). The formation of the heat-precipitated phosphoenzyme is clearly inhibited by ouabain only when the phosphoenzyme is formed in the presence of Mg, Na, and K. Inorganic phosphate decreases the level of the heat-precipitated phosphoenzyme, but not that of the acid-precipitated phosphoenzyme (in the presence of Mg and Na or in the presence of Mg, Na, and K). Moreover, a heat-precipitated, ouabain-sensitive phosphoenzyme forms in the reaction between the eel (Na + K) · ATPase and ³²P_i with or without ATP. The pH stability of the heat-precipitated phosphoenzyme complex is maximal at pH 6 to 8, and this complex shows little or no reactivity with neutral hydroxylamine, suggesting that the phosphate is not bound to an acyl residue of the protein. These experiments indicate that both heat-resistant and acid-resistant phosphoenzymes are formed during the (Na + K) · ATPase reaction at pH 7.4.     

Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

Activation of helper T cells by immune complexes.

Spleen cells from mice primed with dinitrophenylated human γ-globulin (DNP-HGG) did not mount a secondary anti-DNP response in diffusion chamber cultures upon stimulation with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH). The same cells, however, responded to stimulation with DNP-KLH complexed with anti-KLH antibody of rabbit or mouse origin. There is an optimal antigen:antibody ratio at which the immune complexes (IC) must be formed for maximal activity. T cells are required for the immunogenic activity of IC, since T-cell-depleted cultures did not respond. It was found that IC made with carrier and anticarrier antibody stimulated the development of carrier-specific helper T cells in cultures of spleen cells, thymocytes, and nylon wool nonadherent spleen cells from nonimmune mice. In contrast, free carrier did not elicit helper T cells. IC made with carrier and the F(ab′)₂ fragment of anticarrier antibody were immunogenic, but those made with carrier and the Fab′ fragment of anticarrier antibody were not, suggesting that helper T-cell activation is triggered by crosslinking of antigen-specific surface receptors.     

An extracellular fibrillar matrix in gastrulating sea urchin embryos

Fine structural studies of fractured developing sea urchin embryos revealed the existence of a voluminous, fibrillar, extracellular matrix composed of fine filaments, twisting fibers and granules lining the blastocoel of midgastrula embryos. Glycine disaggregated embryos also exhibited this material. The fibrillar matrix is closely associated with the basal lamina of the ectodermal cells of the embryo and histochemical studies suggest it is composed mostly of sulfated glycosaminoglycans. The position of the matrix within the blastocoel as well as its organized association with embryonic cell surfaces is consistent with the hypothesis that it plays a major role in guiding the invaginating archenteron during gastrulation.     

Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo³¹P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

Development and differentiation of mouse blastocysts in serum-free medium

Numerous studies have shown that the in vitro development and differentiation of mouse blastocysts require serum, but the number and nature of serum factors involved remains unclear. In this article, we describe a culture medium, EM-2, containing as a source of protein only commercially purified bovine serum albumin (BSA) and fetuin. This medium supports hatching, attachment and outgrowth of mouse blastocysts. Although attachment and outgrowth are delayed in EM-2 medium 12–15 and 5–8 h, respectively, these events occur at frequencies comparable to those observed in serum-containing media. Trophoblast cells are capable of differentiating in this medium: they synthesize Δ⁵,3β-hydroxysteroid dehydrogenase and their nuclei become polyploid. The inner cell mass also appears to differentiate to some extent in EM-2 medium as evidenced by the appearance of cells with characteristics of parietal endoderm. The fetuin factor is necessary at least for trophoblast outgrowth and the albumin factor is required for the survival and/or growth of the inner cell mass. It is, however, not evident from these studies whether the serum fractions used are actually involved in the induction of differentiation, or whether the early differentiative steps in the mouse blastocyst are preprogrammed and require for expression only a normal cellular metabolic rate.     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

The effects of L-2,4-diaminobutyric acid on the uptake of gamma-aminobutyric acid by a synaptosomal fraction from rat brain

l-2,4-diaminobutyric acid was studied as an inhibitor of gamma-aminobutyric acid uptake by a synaptosomal fraction isolated from rat brain. Competitive inhibition was observed during short-term exposure of the synaptosomal fraction to the inhibitor but noncompetitive inhibition was observed following prolonged exposure. Studies on the mode of action of l-2,4-diaminobutyric acid showed that the synaptosomal fraction was capable of accumulating this compound and that both the uptake and the effectiveness of the inhibitor were sodium-dependent and temperature-sensitive. In addition, the degree of inhibition of gamma-aminobutyric acid uptake was related to the amount of l-2,4-diaminobutyric acid accumulated. It is suggested that the observed noncompetitive inhibition of gamma-aminobutyric acid uptake by l-2,4-diaminobutyric acid is a result of the accumulation of the inhibitor which exerts its effect from within the synaptosomes. Raising the external concentration of gamma-aminobutyric acid to saturating levels did not completely inhibit the accumulation of l-2,4-diaminobutyric acid. Thus, the transport of l-2,4-diaminobutyric acid appears to be mediated, at least in part, by a carrier which is not involved in the transport of gamma-amiuobutyric acid.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Activation of aflatoxin B1 by primary cultures of adult rat hepatocytes: effects of hepatocyte density

Primary monolayer cultures of adult rat hepatocytes readily activate aflatoxin B1 as determined by bacterial mutagenesis (Ames test) and the extent of apparent covalent binding of aflatoxin B1 residues to hepatocyte macromolecules. For intact cultures inoculated with 3 X 10(5)-3 X 10(6) cells/dish, the efficiency of activation decreases with increasing cell density whereas permeabilized hepatocytes prepared from similarly-handled monolayer cultures show with increasing protein proportional increases in the capacity to activate aflatoxin B1. The density effect observed with intact cultured hepatocytes appears not to be due to substrate (aflatoxin B1) or oxygen depletion. These findings have apparent relevance to studies of carcinogen metabolism and in the design of carcinogen/mutagen testing protocols which utilize cultured hepatocytes.     

Structure and genomic organization of the mouse dihydrofolate reductase gene

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5′ untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.