Cytoplasmic Preparations Research Articles

Effect of leucine enkephalin on ethanol produced behavioral depression in the mouse

The effect of leucine enkephalin (LNK) on spontaneous locomotor activity was studied in mice of both sexes. The effect of a depressant dose of ethanol (ET) on LNK-mediated response on motility was also studied. In addition, the determination of specific activities of the hepatic enzymes primarily involved in the metabolism of ET and acetaldehyde was studied. Lactate dehydrogenase (LDH) isoenzymes were also assayed in both plasma and heart tissues. Intraperitoneal injection of LNK, 100 mg/kg, exerted behavioral depression in the male but not female mouse compared to controls. This effect was apparent for the initial 30 min posttreatment. Injection of two smaller doses of LNK, which were devoid of effect on mouse motility, prior to a depressant dose of ET counteracted ET-caused depression of motility only in the female mouse. This became apparent 30 min postdrug injection and lasted for 60 min thereafter. The LNK and ET treatment inhibited only male mouse liver aldehyde dehydrogenase in both cytoplasmic and mitochondrial preparations concomitant with reduction of plasma but not heart LDH 1 isoenzyme from corresponding controls. The results suggest that LNK and ET affect different systems involved in behavioral depression tested and show a potential for LNK in antagonizing the ET effect studied. The data also indicate a sex-dependent effect of LNK on motility and of its interaction with ethanol on the enzymes studied.

Investigations into the thermotrofic phase behaviour of natural membranes extracted from gram-negative bacteria and artificial membrane systems made from lipopolysaccharides and free lipid A

It is reported on investigations into the thermotropic phase behaviour of outer, cytoplasmic, and cell envelope preparations of Gram-negative bacteria, and their amphiphatic compounds - lipopolysaccherides and phospholipids - applying calorimetric, infrared spectroscopic, and 90°-light scattering techniques. Except for free lipid A, the lipid moiety of lipopolysaccharide, all investigated compounds exhibited gel-liquid crystalline phase transitions of the hydrocarbon chains in the temperature range 10 to 42 °C. The transition regions of the outer membrane end lipopolysaccharide preparations are narrower and appear at significantly higher temperatures (around 37 °C) as those from the cytoplasmic membrane and phospholipid preparations (around 26 °C). For both amphiphatic compounds the phase transitions are shifted to lower values at reduced growth temperature of the cells.

Electrophoretic and serological analyses of cytoplasmic antigens fromAspergillus fumigatusduring growth of conidia to mature mycelia

The changes of cytoplasmic components concomitant with conidium to mature mycelium growth of Aspergillus fumigatus strain Ag 507 were analysed by one- and two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE; 2-DE). SDS-PAGE monitored molecular weight differences between components of cytosol preparations obtained from conidia and those through 96 h of mycelial growth. 2-DE analyses indicated that some components characteristic of mature cytosol begin to appear by 7 h. Cytoplasmic preparations absorbed with rabbit immunoglobulins raised to mature cytosol were analysed by 2-DE. Conidia cytosol components were not absorbed to a great degree, unlike those from later stages of mycelial growth, which indicates that cytosol components may be changed and/or synthesized de novo during growth of the fungus. Analysis of the cytosol preparations by fused rocket immunoelectrophoresis showed that some components are synthesized in different amounts at various times during growth: 3, 4, 7, 8, and 18 h of growth, components begin to appear that may be synthesized de novo. Enzyme-linked immunosorbent assay with rabbit antiserum to mature cytosol and cytosol preparations obtained from conidia through 96 h of growth, indicated differences of molecular structures between the cytosol preparations. The anticytosol IgG and IgE titers of sera from patients with allergic bronchopulmonary aspergillosis were both elevated and fluctuated with each preparation. The specific IgG and IgE titers both appeared to be elevated with cytosol preparations obtained from 4, 5, 7, and 9 h of growth and highest against the 96 h preparation.

Retinoic acid-binding protein in the axolotl: Distribution in mature tissues and time of appearance during limb regeneration

Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all- trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all- trans and 13- cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [ 3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb.

Estradiol-17 beta affects estrogen receptor distribution and elevates progesterone receptor content in baboon aorta.

We used the synthetic estrogen R2858 (moxestrol) and estradiol-17 beta, respectively, to characterize the estrogen receptor in baboon (Papio sp.) aortic or myocardial cytoplasmic and nuclear preparations. We observed regional differences in the cytoplasmic fraction estrogen and progesterone receptor content of aortic arch, thoracic aorta, and abdominal aorta when tissues from either oophorectomized or oophorectomized estradiol-17 beta-treated subjects were compared. The estrogen receptor content was highest in the abdominal aorta and lowest in the aortic arch. In contrast, the cytoplasmic fraction progesterone receptor content was highest in the aortic arch and lowest in the abdominal aorta. The nuclear fraction estrogen receptor could not be demonstrated in preparations from cardiovasculature of oophorectomized female baboons. The use of Silastic implants to administer a physiologic concentration of estradiol-17 beta to oophorectomized female baboons caused a 20% to 50% reduction in cytoplasmic fraction estrogen receptor content, which was quantitatively accounted for by the appearance of estrogen receptor in the corresponding nuclear aortic or myocardial preparation. Estrogen administration caused a 20% to 40% increase in cytoplasmic fraction progesterone receptor content in both myocardium and aorta; however, differences were significant only for abdominal aorta (p less than 0.05). Estradiol-17 beta treatment caused a tenfold increase in uterine cytoplasmic fraction progesterone receptor content in treated as compared to oophorectomized control females, suggesting that baboon cardiovasculature is less sensitive to changes in endogenous estrogen concentration than is uterus. The ability of estradiol-17 beta to affect apparent intracellular distribution of baboon cardiovascular estrogen receptors and to elevate cytoplasmic fraction progesterone receptor content suggests that these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate primate cardiovascular cell function.

Specific postsynaptic density proteins bind tubulin and calmodulin-dependent protein kinase type II.

Cytoskeletal interactions which contribute to the assembly of the postsynaptic density (PSD) were investigated. PSDs bound 125I-tubulin specifically with an apparent Km of 2 X 10(-7) M and a Bmax of about 1 nmol/mg of protein. 125I-Tubulin blots revealed that a group of polypeptides between Mr 135,000 and 147,000 (P-140) was a major tubulin-binding PSD component. The P-140 polypeptides were highly enriched in the PSD fraction of purified synaptosomes and could not be detected in crude brain cytoplasm preparations. These polypeptides were subject to phosphorylation by endogenous calmodulin-dependent protein kinase type II, with a concomitant reduction in 125I-tubulin binding. The tubulin-binding polypeptides could also associate with the radiolabeled alpha- and beta-subunits of the calmodulin-dependent protein kinase. These observations are consistent with a role for the P-140 polypeptides in organizing the molecular structure of the PSD. The data also suggest that this structure may be modified by Ca2+-sensitive phosphorylation, thus permitting neuronal activity to modulate the cytoskeletal interactions of the PSD.

13] Purification of the aa3-type cytochrome-c oxidase from Rhodopseudomonas sphaeroides

This chapter describes the purification of the aa 3 -type cytochrome-c oxidase from Rhodopseudomonas sphaeroides . The chapter outlines the procedures for the preparation of the bacterial membranes and purification of the oxidase. R. sphaeroides is a nonsulfur bacterium that, when grown anaerobically in the light, develops a photosynthetic apparatus. In the dark and in the presence of oxygen, the photosynthetic apparatus is suppressed and the bacterium utilizes a respiratory chain. The cytochrome-c oxidase from R. sphaeroides can easily and quickly be prepared in small amounts. The enzyme is immunologically related to that found both in yeast mitochondria and P. denitrificans. This oxidase appears to be structurally similar to other bacterial aa 3 -type oxidases, though does not function as a proton pump. The success of the purification of the oxidase critically depends on the purity of the cytoplasmic membrane preparation.

Estrogen-mediated cytoplasmic and nuclear distribution of rat cardiovascular estrogen receptors.

We used either the synthetic estrogen R2858 (moxestrol) or estradiol-17 beta to characterize estrogen receptors in cytoplasmic (R2858) and nuclear (estradiol-17 beta) preparations from rat aorta and myocardium. Relative steroid specificity studies showed that only estrogens were effective inhibitors of R2858 or estradiol-17 beta binding to aortic and myocardial estrogen receptors, whereas androgens, progestins, and cortisol were ineffective inhibitors. Low ionic strength sucrose density gradient analyses showed that myocardial estrogen receptors that localized in the cytoplasmic fraction migrated as macromolecules with sedimentation coefficients of 8S to 9S. In contrast, two binding components of sedimentation coefficients 8S to 9S and 10S to 11S were characteristic of the estrogen receptors localized in aortic cytoplasmic preparations. High ionic strength sucrose density gradient analysis showed that aortic and myocardial estrogen receptors localized in the nuclear fraction migrated as macromolecules with sedimentation coefficients of 4S to 6S. Saturation analyses showed that aortic and myocardial cytoplasmic preparations from intact young mature male rats contained 50.6 +/- 12.9 (mean +/- SD) and 51.0 +/- 14.1 fmol receptor/mg DNA, respectively. The respective R2858 dissociation constants were 0.42 and 0.15 nM. Estrogen receptors could not be demonstrated in nuclear preparations from cardiovasculature of intact males. Estradiol-17 beta injection of intact young mature male rats caused "depletion" of aortic and myocardial cytoplasmic fraction estrogen receptors and resulted in the appearance of 51.9 +/- 21.0 and 36.9 +/- 9.5 fmol receptor/mg DNA in the corresponding nuclear fractions. The respective estradiol-17 beta dissociation constants were 1.56 and 0.71 nM. Increased estrogen receptor content of cardiovascular nuclear fractions of estradiol-17 beta injected male rats correlated well with the concomitant decreased cytoplasmic fraction receptor content. The ability of estradiol-17 beta to affect localization of cardiovascular estrogen receptors between cytoplasmic and nuclear fractions suggests these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate cardiovascular cell function.

Androgen directs apparent cytoplasmic and nuclear distribution of rat cardiovascular androgen receptors.

We used either the synthetic androgen R1881 (methyltrienolone) or 5 alpha-dihydrotestosterone (5 alpha-DHT) to characterize androgen receptors in rat aortic and myocardial cytoplasmic and nuclear preparations. Relative steroid specificity studies established that only androgens were effective inhibitors of R1881 (cytoplasmic) or 5 alpha-DHT (nuclear) binding to aortic and myocardial androgen receptors, whereas estrogens, progestins, and cortisol were ineffective inhibitors. Low ionic strength sucrose density gradient centrifugation analyses showed that androgen receptors in aortic and myocardial cytoplasmic preparations migrated as macromolecules with sedimentation coefficients of 8 to 9S, whereas androgen receptors in aortic and myocardial nuclear preparations migrated as macromolecules with sedimentation coefficients of 4 to 5S (high ionic strength buffer). Saturation analyses established that aortic and myocardial cytoplasmic preparations from intact, untreated young mature female rats contained 45.8 +/- 9.7 (mean +/- SD) and 63.3 +/- 20.7 fmol androgen receptor/mg DNA, respectively. The respective R1881 dissociation constants were 0.60 and 0.32 nM. Androgen receptors could not be demonstrated in nuclear preparations from the cardiovasculature of intact females. Testosterone injection of intact young mature female rats caused apparent depletion of androgen receptors in aortic and myocardial cytoplasmic preparations and resulted in concomitant appearance of 57.1 +/- 22.2 and 52.3 +/- 21.5 fmol receptor/mg DNA in the corresponding nuclear preparations. The respective 5 alpha-DHT dissociation constants were 4.46 and 1.63 nM. The ability of testosterone to affect apparent intracellular distribution of cardiovascular androgen receptors suggests that the receptors are physiologically functional and indicates that androgen may directly regulate cardiovascular cell function.

The expression of the SV40 early region in the transformed human cell line SV80

The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line.

Promotion of Septation in Irradiated Escherichia coli by a Cytoplasmic Membrane Preparation

Septation can be promoted in an X-irradiated lon mutant of Escherichia coli K-12 by the addition of an E. coli B/r cytoplasmic membrane preparation to the postirradiation plating medium. The promotion of septation was not associated with an inhibition of growth rate. Two distinct cytoplasmic membrane-associated properties were necessary to promote septation. One of these, the cytochrome-based electron transport system, produced anaerobic conditions by the reduction of oxygen dissolved in the medium. The second system, functioning independently from the first, altered substances found in the peptone and yeast extract components of the postirradiation plating medium. When both systems were operative, significant repair of the cell division mechanism occurred.

A detailed investigation of the cytoplasmic cortisol-binding receptor of North American eel (Anguilla rostrata) tissues

The in vitro binding of tritiated cortisol to ammonium sulfate precipitate (35% saturation) prepared from the gill and gut mucosal cytosol of the North American eel (Anguilla rostrata) was investigated. The sodium molybdate stabilized cytoplasmic preparations bound tritiated cortisol with the following parameters: gill, equilibrium dissociation constant (KD) = 3.7 ± 0.4 nM, (± SEM; n = 4), the maximum concentration of binding sites (Nmax) = 294 ± 26 fmol/mg protein; gut, KD = 5.2 ± 0.4 nM, Nmax = 1085 ± 288 fmol/mg protein. The [3H]cortisol–receptor complexes sedimented on linear (16–41% w/v) glycerol density gradients in single peaks at 6.7S–7.0S or 3.0S–3.6S in hypo- or hyper-tonic (± 0.4 M KCl) gradients, respectively. Sephacryl S-300 column chromatography of the hormone–receptor complex yielded the following hydrodynamic parameters: gill, relative mass (Mr) = 292 000 daltons, Stokes radius (Rs) = 78.7 Å(1 Å = 0.1 nm), frictional ratio (f/f0) = 1.79; gut, Mr = 242 000 daltons, Rs = 68.8 Å, f/f0 = 1.66. Competition studies revealed the following competitive hierarchies of radioinert steroids vis-à-vis the inhibition of [3H]cortisol binding to the receptor with both tissues: cortisol > 11-deoxycortisol > 21-deoxycortisol > 17α-hydroxyprogesterone [Formula: see text] corticosterone [Formula: see text] 11-deoxycorticosterone > 11β-hydroxyprogesterone. Aldosterone, cortisone, progesterone, or promegestone (R5020) hardly competed. These findings underline the importance of the C-17, C-21, and C-11 hydroxyl groups in receptor binding. Hydroxylation of progesterone in positions C-17, C-21, and C-11 contributed free energy changes (ΔG) of −5.8 to −6.2, −3.1 to −3.9, and −1.3 kJ/mol, respectively, to the binding of steroids to the eel glucocorticoid receptor. From these data we conclude that the piscine cortisol receptor is different from other vertebrate glucocorticoid receptors because of its physical and thermodynamic characteristics and its function in mediating electrolyte homeostatic action of a typical glucocorticoid in the transport epithelia. It is conceivable that the fish glucocorticoid receptor is an ancestral form of the glucocorticoid and (or) mineralocorticoid receptors of vertebrates.

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at −196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ƒ, cytochrome b-559 and cytochrome b 6, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at −196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.

Effects of Ca 2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 m M EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca 2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca 2+ ion levels are kept low, but when Ca 2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca 2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.

Separation and characterization of receptor-translocation inhibitors from AH 130 tumor cells

The objective of this investigation was to study the relationship between glucocorticoid resistance and macromolecular receptor-translocation inhibitors (MTIs). MTIs in various cytoplasmic preparations are known to inhibit the “activated” receptor-steroid complex association with isolated nuclei, chromatin, or DNA. It was found that the MTI in the cytosol of AH 130 tumor cells (glucocorticoid resistant cells) appeared to be about 5 times more inhibitory than crude MTI from rat liver. Another difference between these MTI preparations was that ATP decreased the inhibition by crude MTI from rat liver, but had little effect on that of MTI from the tumor cells. Both preparations gave three fractions of material with inhibitory activity on DEAE-cellulose chromatography. The first fraction (Peak I), eluted with about 0. l M NaCl, was the largest fraction separated from the tumor cytosol, but a minor fraction ofthat from liver. In the presence of 5 mM ATP, Peak I from rat liver enhanced nuclear binding, but that from the tumor did not, suggesting that these fractions were qualitatively different. The other two fractions (Peak II and Peak III), eluted with about 0.2 M and 0.3 M NaCl, respectively, were comparable in the two preparations.

Comparison of polyadenylic acid and oligouridylic acid messenger RNA associated proteins

Various subclasses of messenger ribonucleoprotein particles were prepared from free cytoplasmic and polysome fractions of rat liver on the basis of the homopolymeric content of the constituent RNA's. Two major proteins were evident in the free cytoplasmic preparations: the poly(A)-binding protein was the major constituent of polyadenylated components and a 60 kilodalton protein was the major protein in oligouridylated components. In addition to the poly(A)-binding protein, the polysome fractions contained a 74 kilodalton protein that was present in all subclasses of particles. With both free cytoplasmic and polysome preparations, chromatography on columns of poly(U)-sepharose separated poly-adenylated mRNP's largely on the basis of the length of the poly(A) tract; mRNP's containing short poly(A) tracts (fragment distribution centered on 34 residues) were not retained by the columns, presumably because of the interaction of the poly(A) with poly(A)-binding protein.

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.

Regulation of cytoplasmic mRNA prevalence in sea urchin embryos: Rates of appearance and turnover for specific sequences

Complementary DNA clones representing cytoplasmic poly(A) RNAs of sea urchin embryos were hybridized with metabolically labeled cytoplasmic RNA preparations and the rates of appearance and of decay for each transcript species were determined at the blastula-gastrula stage of development. The prevalence of the transcripts chosen for this study ranged, on average, from about one molecule per cell to a few hundred molecules per cell. The embryos were labeled continuously for 18 hours with [ 3H]guanosine, beginning at 24 hours post-fertilization. The amount of cytoplasmic [ 3H]poly(A) RNA that hybridized to each cloned sequence was determined and the specific activity of the [ 3H]GTP pool was measured in the same embryos. Rate constants for the entry of each transcript species into the cytoplasm, and for its decay were extracted from these data. The embryo transcript species identified by the cloned probes displayed a range of stabilities. Half-lives of only a few hours were measured both for a very rare sequence and for a moderately prevalent sequence. Other newly synthesized transcripts, including sequences that first appear during embryonic development, as well as sequences also represented in maternal RNA, are far more stable. We conclude that cytoplasmic RNA turnover rate is a major variable in the determination of the cytoplasmic level of expression of embryo genes. The entry rates of the transcripts into the cytoplasm also varied, from a few molecules per embryo per minute to several hundred, depending on the sequence. By comparing the mass of transcripts of a given sequence in the embryo to the mass of transcripts of that sequence accumulating as a result of new synthesis, the point at which embryo transcription accounts for the major fraction of the cytoplasmic molecules could be estimated. This calculation showed that for some sequences maternal transcripts persist well beyond gastrulation, while other embryo poly(A) RNA species are largely the product of transcription in the embryo nuclei from the blastula stage onwards. There is no single stage at which all maternal transcripts are suddenly replaced by newly synthesized embryo transcripts. Primary transcription rates were measured for two sequences by determining accumulation of label in these RNA species soon after addition of [ 3H]guanosine to the cultures. Comparing these rates to the cytoplasmic entry rates, we did not detect a significantly greater nuclear transcription of the sequence homologous to the cloned probe.

Facilitation of Skin Allograft Survival by Blood Leucocyte Extracts

Fresh and frozen-stored mitochondrial, microsomal, and endoplasmic reticulum extracts of blood leucocytes act as potent alloantigens in human recipients. Similar results were obtained with freshly prepared extracts consisting of mixtures of all cytoplasmic fractions; storage of such mixtures at--20 C, followed by 1-2 hr thawing at 37 C abrogated their capacity to induce allograft sensitivity in 45 of 47 recipients. Donor-specific skin allografts and grafts from other sources exhibited significant attenuations in the tempo and intensity of rejection, ranging from first-set rejection to chronic rejection and/or to prolongations in allograft survival. In contrast with the 66.2% rejection rate of first-set skin grafts at 10 days in 71 normal subjects, only 29% of skin grafts from the leucocyte donor applied to 24 recipients of 0.1 to 9 Transplantation Antigen (T.A.) units of pooled cytoplasmic mixtures were rejected by that time. Only 17.4% similar grafts in 23 recipients of 25 to 515 T.A. units were rejected at 10 days. Seventeen skin grafts placed on recipients of 45 to 140 T.A. units of the same cytoplasmic preparation exhibited a slow rejection characterized by progressive shrinkage and eventual disappearance, with no evidence of hemorrhagic necrosis (chronic skin graft rejection). These results support the possibility that the attenuations in allograft reactivity observed in patients with end-stage renal disease after blood transfusions may be related to the leucocyte components of such transfusions. The capacity of blood transfusions to decrease reactivity to renal allografts in uremic patients maintained on hemodialysis stands in contrast with the ability of such transfusions to sensitize normal human recipients to donor-specific skin allografts. The differential effect may be related to the immunosuppressed state(s) documented in the uremic population, and/or the use of immunosuppressive drug therapy in such patients at the time of transplantation and thereafter.

The germinal vesicle material required for sperm pronuclear formation is located in the soluble fraction of egg cytoplasm.

The chromatin of Xenopus laevis sperm nuclei was induced to decondense, swell and form mitotic chromosomes following its injection into mature Rana pipiens oocytes. In contrast, the sperm chromatin did not decondense or form mitotic chromosomes when injected into oocytes from which the germinal vesicle (GV) was removed prior to the initiation of maturation. Injection into enucleated oocytes of the material extracted from manually-isolated GVs restored their ability to decondense sperm nuclei. This soluble GV material was stable at 18 degrees C for 16 h but was inactivated by heating to 80 degrees C for 10 min. We examined the distribution of this GV material in a cytoplasmic preparation from activated eggs which can induce sperm pronuclear formation in vitro. The cytoplasmic preparation was separated into soluble and particulate fractions by centrifugation and then each fraction was injected into enucleated eggs to determine whether or not it restored the ability to decondense sperm nuclei. We found that the soluble, but not the particulate fraction could restore the ability to decondense sperm nuclei to enucleated oocytes. This result clearly indicates that the soluble fraction contains most of the GV material required for chromatin decondensation. However, since the soluble fraction fails to decondense sperm chromatin in vitro in the absence of material from the particulate fraction, sperm pronuclear formation appears to require both the soluble material derived from the GV and particulate material which can develop in the oocyte cytoplasm in the absence of the GV.