Wt Fraction Research Articles

Constituents of cigarette smoke inhibit human granulosa cell aromatase

Recent epidemiologic studies suggest that women smokers have lower endogenous estrogen levels than nonsmokers. The effects of aqueous extracts of cigarette smoke on aromatase were evaluated in cultures of human granulosa cells. Aqueous extracts of cigarette smoke inhibited the conversion of androstenedione (delta 4A) to estradiol in a dose-dependent manner. Dialysis experiments demonstrated that 90% of the inhibitory activity of aqueous extracts of cigarette smoke was in the less than 1000 mol wt fraction. Removal of the aqueous extract of cigarette smoke from the culture medium resulted in a complete reversal of the inhibition of delta 4A aromatization. Addition of supraphysiologic concentrations of delta 4A (73 microM) to the culture medium blocked the smoke-induced inhibition of aromatization. Two low-molecular-weight components of cigarette smoke, nicotine and anabasine, inhibited granulosa cell aromatase in a dose-dependent manner. These studies suggest that constituents of cigarette smoke inhibit a major steroidogenic pathway.

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium.

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.

Inhibition of luteinizing hormone/human chorionic gonadotropin-stimulable adenylyl cyclase activity in rat luteal membranes by nonsteroidal component(s) in human follicular fluid.

We examined the ability of nonsteroidal components of human follicular fluid (hFF) to alter gonadotropin responsiveness using the LH/hCG-sensitive adenylyl cyclase system of rat luteal membranes. Follicular aspirates were obtained from regularly ovulatory women (n = 10) whose follicles were stimulated by human menopausal gonadotropin and hCG as part of an in vitro fertilization program. hFF from large follicles was pooled and extracted with 10% (wt/vol) activated charcoal. Maximal hCG stimulation of adenylyl cyclase activity obtained with 10 micrograms/ml hCG and 100 microM of the hydrolysis-resistant GTP analog guanyl 5'-yl-imidodiphosphate was significantly inhibited by hFF in a dose-dependent manner. Addition of about 500 micrograms hFF protein caused inhibition of 70% compared to the control value. Fractionations of hFF by ultrafiltration using membranes of precalibrated pore size demonstrated that the inhibitory activity was associated with a less than 10,000 mol wt fraction; 3 micrograms protein/assay of this fraction resulted in 50% inhibition (IC50) of maximal hCG stimulation. The inhibitory activity also passed through an Amicon YM-2 membrane (mol wt retention, 1,000), but not through an Amicon YC-05 membrane (mol wt retention, 500). An IC50 of about 0.01 microgram protein/assay was found for both the 500-1,000 and the 1,000-5,000 mol wt fractions. NaF or forskolin-stimulated adenylyl cyclase activity was not altered by unfractionated hFF or by the 500-10,000 mol wt subfractions, suggesting that inhibition was limited to LH/hCG stimulation. Further analysis of the effects of low mol wt fraction on hCG stimulation of adenylyl cyclase indicated that enzyme inhibition was not accompanied by a shift in the hCG concentration required for half-maximal stimulation (the apparent activation constant) compared to dose-response curves obtained in the absence of added fraction. Equilibrium binding studies showed that [125I]hCG interaction with luteal membranes was significantly inhibited by hFF; 7 micrograms protein/assay of the less than 10,000 mol wt fraction reduced specific binding by 60%. Moreover, kinetic analysis carried out in the absence or presence of a fixed amount of low mol wt fractions revealed a competitive type of binding inhibition. Our data demonstrate that a nonsteroidal component(s) of hFF has a direct inhibitory effect on LH/hCG-responsive luteal adenylyl cyclase and that the inhibitor(s) exerts its actions through a mechanism involving competition with LH/hCG for the same binding sites.

Electrophoretic characterisation of low‐molecular weight wheat protein of variable solubility

AbstractA low mol. wt protein (S protein) which has a high affinity for endogenous polar lipid was isolated from wheat gluten, partially purified and compared electrophoretically with low mol. wt components of several previously studied wheat protein preparations. Mobilities of S protein components in acidic polyacrylamide gel electrophoresis (PAGE) were very close to those of the chloroform/methanol soluble low mol. wt proteins (CM proteins). A fraction of S protein separated by gel filtration chromatography on Sephadex G‐50 (S‐III protein) was almost identical to CM proteins by two‐dimensional electrophoresis. Bands with mobilities similar to those of S‐III protein were present in PAGE patterns of gliadin, the low mol. wt fractions IV and V obtained from gliadin by gel filtration chromatography on Sephadex G‐200, and the albumin/globulin fraction of flour prepared by the Osborne solubility fractionation. Because of its variable solubility S protein cannot be unambiguously classified according to the Osborne solubility classification of plant proteins.

Reduced food intake following intracerebroventricular administration of a low molecular weight fraction of plasma from free-feeding fowl.

Plasma was collected from free-feeding Leghorn cockerels and partitioned by gel filtration into fractions of different molecular weight ranges. The individual fractions were then lyophilized and reconstituted to four times the original concentration. The plasma was administered to 10-week-old Leghorn cockerels via a stainless steel guide cannula, stereotaxically implanted into the lateral cerebral ventricle. Sated cockerels received 10 microliters intracerebroventricular (ICV) injections of one of the concentrated plasma fractions or of a control injection of artificial cerebrospinal fluid. Food and water intake were monitored following injection. Food intake was significantly decreased by the ICV injection of plasma fractions less than 1500 mol wt, whereas water consumption was not significantly different from that of the controls. The 1500 to 5000 mol wt fraction and the fraction above 5000 did not alter either food or water intake. These results suggest that the plasma of free-feeding domestic fowl contains a low molecular weight factor that is involved in the regulation of food intake.

Immunological responses to protein, carbohydrate and lipid fractions of Nocardia asteroides in mice.

The protein, polysaccharide and phospholipid constituents of Nocardia asteroides have been partially purified and their immunogenicity studied in mice. Humoral and cellular immune responses were demonstrated against a crude cytoplasmic protein fraction (CPF). Two fractions of CPF were prepared on a Sephadex G-200 column; a high mol. wt fraction, fraction-1(F1) was capable of eliciting both types of immune responses, whereas fraction-2(F2) behaved more like a hapten. Phosphatides elicited only humoral responses whereas polysaccharides were non-immunogenic.

Interleukin 2 production in B cell chronic lymphocytic leukemia

Interleukin 2 (IL 2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was investigated in 22 patients with active untreated B cell chronic lymphocytic leukemia (B- CLL) and in 15 healthy donors. PBMCs from healthy donors demonstrated an IL 2 synthesis of 12.4 +/- 10 U/mL. B-CLL PBMCs produced a significant amount of IL 2 (8 +/- 6.6 U/mL) despite the low percentage of T cells (13% +/- 8%) associated with this disease compared with that found in healthy donors (63% +/- 7.5%). If IL 2 production is expressed as units per milliliter per 10(4) T cells, its level in patients with B- CLL (1.1 U/mL/10(4) T cells) is five times greater than that of the controls (0.19 units). When expressed as units per milliliter per liter of blood, the B-CLL patients produce approximately 12 times as much IL 2 as controls. IL 2 production in normal controls was doubled after irradiation of PBMCs or addition of indomethacin. This increase was not seen with B-CLL PBMCs suggesting that the latter have been devoid of prostaglandin-producing normal IL 2 suppressor cells. By mixing normal or B-CLL T cells with non-T cells we found that T cells from patients with B-CLL stimulated by normal accessory cells produced the same amount of IL 2 as normal T cells. Moreover, B-CLL non-T cells (mainly B leukemic cells) produced no IL 2 themselves but played a much more efficient role in IL 2 production than did non-T cells from healthy donors. This was not due to detectable IL 1 production by these cells. The IL 2 produced by B-CLL PBMCs was partially purified and recovered in a 16,000 mol wt fraction, the same mol wt as IL 2 from normal cells.

Interleukin 2 Production in B Cell Chronic Lymphocytic Leukemia

Interleukin 2 (IL 2) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) was investigated in 22 patients with active untreated B cell chronic lymphocytic leukemia (B-CLL) and in 15 healthy donors. PBMCs from healthy donors demonstrated an IL 2 synthesis of 12.4 +/- 10 U/mL. B-CLL PBMCs produced a significant amount of IL 2 (8 +/- 6.6 U/mL) despite the low percentage of T cells (13% +/- 8%) associated with this disease compared with that found in healthy donors (63% +/- 7.5%). If IL 2 production is expressed as units per milliliter per 10(4) T cells, its level in patients with B-CLL (1.1 U/mL/10(4) T cells) is five times greater than that of the controls (0.19 units). When expressed as units per milliliter per liter of blood, the B-CLL patients produce approximately 12 times as much IL 2 as controls. IL 2 production in normal controls was doubled after irradiation of PBMCs or addition of indomethacin. This increase was not seen with B-CLL PBMCs suggesting that the latter have been devoid of prostaglandin-producing normal IL 2 suppressor cells. By mixing normal or B-CLL T cells with non-T cells we found that T cells from patients with B-CLL stimulated by normal accessory cells produced the same amount of IL 2 as normal T cells. Moreover, B-CLL non-T cells (mainly B leukemic cells) produced no IL 2 themselves but played a much more efficient role in IL 2 production than did non-T cells from healthy donors. This was not due to detectable IL 1 production by these cells. The IL 2 produced by B-CLL PBMCs was partially purified and recovered in a 16,000 mol wt fraction, the same mol wt as IL 2 from normal cells.

Cytosolic cofactors and dihydrolipoamide stimulate hepatic microsomal 5'-deiodination.

The effect of rat hepatic cytosolic constituents on microsomal 5'-deiodinase (5'-DI) activity in the presence of the dihydrolipoamide-generating system or dihydrolipoamide itself has been studied. Reconstitution experiments have demonstrated for the first time the essential role of cytosol as well as lipoamide and NADH in the activation of 5'-iodothyronine DI to a level equivalent to that attained in the presence of 5 mM dithiothreitol. Omission of NADH, lipoamide, or cytosol reduced the DI activity by 86%. By gel filtration chromatography of the cytosol, a fraction of intermediate mol wt (approximately 13,000) was shown to be highly stimulatory of microsomal DI in washed microsomes, provided either a dihydrolipoamide-generating system (lipoamide dehydrogenase, as well as NADH and lipoamide) or dihydrolipoamide alone was added. These observations suggest a possible role for a heretofore unreported reducing system influencing hepatic 5'-deiodination, wherein cytosolic cofactors appear to function as efficient intermediaries which enhance the action of a dihydrolipoamide in the activation of isolated hepatic microsomal 5'-DI.

Lactogen receptors in rat Leydig cells: analysis of their structure with bifunctional cross-linking reagents.

[125I]Iodohuman GH was found to bind to receptors with specificity for lactogenic hormones in a Triton X-100 extract from Leydig cell membranes displaying an affinity constant of 3.8 X 10(9) M-1 and a binding capacity of 167 fmol/mg protein. Cross-linking of solubilized [125I]iodohuman GH-receptor complexes with disuccinimidyl suberate followed by analysis by sodium dodecyl sulfate-gel electrophoresis in the presence of beta-mercaptoethanol and autoradiography resulted in the appearance of bands with apparent mol wt of 113,000, 103,000, 59,000, and 53,000. The appearance of these bands was prevented by incubation in the presence of lactogenic hormones. By using a two-dimensional electrophoresis technique (first dimension under nonreducing conditions; second dimension under reducing conditions), it was demonstrated that a fraction of the mol wt 59,000 species can be released from the mol wt 103,000 species upon cleavage of disulfide bonds. These results suggest the existence of lactogen receptor species with approximate mol wt of 91,000, 81,000, 37,000, and 31,000 in Triton X-100 extracts from Leydig cell membranes if the contribution of the free hormone (mol wt, 22,000) is subtracted. A fraction of the mol wt 37,000 subunits appears to be contained within the 81,000 species linked through disulfide bonds.

Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin.

We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.

Luteinized rat ovaries contain a sterol carrier protein.

Highly luteinized rat ovaries contain a soluble 13,000 mol wt factor which binds [3H]cholesterol and enhances cholesterol transfer from phospholipid vesicles to mitochondria. The ovarian cholesterol transfer protein was purified 1200-fold. It has a basic isoelectric point, is heat stable, and has an amino acid composition similar to that reported for rat liver sterol carrier protein2. Antisera raised against preparations of rat liver sterol carrier protein2 precipitated labeled cholesterol associated with the ovarian 13,000 mol wt fraction. A line of partial identity was obtained when the ovarian fractions and purified rat liver sterol carrier protein2 were run against these antisera in double immunodiffusion. We conclude that luteinized rat ovaries contain a sterol carrier protein with properties similar to hepatic sterol carrier protein2. This protein may facilitate the intracellular distribution of cholesterol in ovarian cells.

Stimulating effect of MDP and its adjuvant-active analogues on guinea pig fibroblasts for the production of thymocyte-activating factor.

Synthetic muramyl dipeptide (MDP) could stimulate skin fibroblasts of the guinea pig to produce thymocyte-activating factor, which augments the proliferative response of thymocytes to phytohemagglutinin (PHA). Adjuvant-active analogues of MDP also stimulated fibroblasts to produce the factor, whereas adjuvant-inactive analogues failed to do so. Thus a marked parallelism was found between adjuvant activity of these compounds and the stimulating effect on fibroblasts. Thymocyte-activating factor derived from MDP-stimulated fibroblasts was found in the fraction of mol wt 30,000-60,000 by gel filtration on a Sephacryl S-200 column. Furthermore, this factor did not exhibit T cell growth factor activity.

Biochemical analysis of a 40,000 mol. wt mouse serum protein which binds β 2-microglobulin and has serological cross-reactivity with H-2 antigens

A 40,000 (40 K) mol. wt mouse serum protein which binds β 2-microglobulin (β 2M) has been purified to homogeneity by a modification of the procedure of Kvist & Peterson (1978). Biochemistry 17, 4794–4801. After gel filtration, sodium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and affinity chromatography on an anti-H-2 a immunoabsorbent column, four proteins with apparent mol. wts of 12, 40, 55 and 75 K were specifically immunoprecipitated with rabbit anti-H-2 a. These individual proteins were subsequently purified to homogeneity by preparative SDS-PAGE. The identity of the 12 K mol. wt protein was unequivocally established to be mouse β 2M based on amino acid composition, N-terminal analysis and reactivity with rabbit anti-β 2M. The 40 K mol. wt protein was the only protein that bound β 2M since radioimmunoprecipitation analysis of the high mol. wt fraction from fresh serum (with rabbit anti-β 2M) yielded only the 40 K mol. wt protein and β 2M. This result also precluded the possibility of a pre-existing complex of the 55 and 75 K mol. wt proteins with the 40 K mol. wt protein and β 2M. The copurification of the 55 and 75 K mol. wt proteins with the 40 K mol. wt protein and β 2M, was explained on the basis of their immunological cross-reactivity. The 55 and 75 K mol. wt proteins were found to be structurally distinct from the 40 K mol. wt protein as judged by amino acid analysis and peptide mapping. The similarity in amino acid composition of the 40 K mol. wt protein and H-2, in addition pto its cross-reactivity with H-2 xenoantisera, suggested the possibility of their structural relationship. However, the 40 K mol. wt protein differed from H-2 in the following respects: (1) H-2 alloantigenic determinants were not present on the 40 K. mol. wt protein, (2) the 40 K mol. wt proteins from two strains of mice had similar peptide maps indicating the lack of structural polymorphism in these strains, (3) the 40 K mol. wt protein had a more acidic and less restricted p I value than H-2, and (4) no common peptides were found in proteolytic digests of H-2 and the 40 K mol. wt proteins as analysed by two different methods. These results demonstrate that the β 2M-binding murine serum protein is not specifically associated with other proteins, is not generated from a precursor present in the serum and bears no apparent homology to H-2 molecules despite serological cross-reactivity.

ATPase-associated oligomycin resistance in Acanthamoeba castellanii.

The multiplication rate of "wild-type" (WT) populations of Acanthamoeba castellanii was inhibited 50% by approximately 3 microgram oligomycin/ml; OliR2, an oligomycin resistant cell line, required approximately 27 microgram/ml for the same inhibition. ATPase solubilized from OliR2 mitochondrial fractions required 3--10-fold higher concentrations of oligomycin than did identical WT fractions to achieve 50% inhibition of activity. Resistance was correlated with altered mitochondrial ATPase sensitivity to oligomycin.

Inhibition of differentiation of mouse myeloid leukemia cells by heat-stable calf serum components of very high molecular weight

Mouse myeloid leukemia cells (MI) were induced to differentiate into macrophages and granulocytes by various inducers including glucocorticoid. The tumour promoter 12- O-tetradecanoylphorbol-13-acetate (TPA) inhibited the induction of differentiation of Ml cells in medium containing calf serum, but enhanced the induction in medium containing fetal calf serum and several inducers. For elucidation of the factor(s) in serum affecting the response of Ml cells to tumour promoters, calf serum was fractionated by Sephadex G-200 gel filtration. Differentiation of Ml cells induced by dexamethasone was markedly inhibited by TPA and high mol. wt fractions of calf serum eluted in the void volume and low mol. wt fractions that co-migrated with bovine serum albumin. High mol. wt fractions alone inhibited the differentiation of Ml cells induced by dexamethasone, and also acted additively with TPA in inhibiting the differentiation. The inhibition by high mol. wt fractions was not related to cytotoxicity and was reversible. The differentiation of Ml cells induced by proteinous inducer or lipopolysaccharide was also inhibited by high mol. wt fractions. The inhibitory factor was heat stable (70°C for 20 min or 90°C for 10 min). These results suggest that the tumour promoter and calf serum components cooperate in inhibiting differentiation of mouse myeloid leukemia cells induced by various inducers.

Peroxidase antimicrobial system of human saliva: requirements for accumulation of hypothiocyanite.

Human saliva was fractionated to determine the components required for production and accumulation of the antimicrobial oxidizing agent, hypothiocyanite ion (OSCN-). The required components were: 1) peroxidase activity and thiocyanate ion (SCN-), 2) the saliva sediment, which produced hydrogen peroxide (H2O2) in the presence of oxygen and a divalent cation, and 3) heat-stable factors of the saliva supernatant. The supernatant factors were separated into high- and low-mol wt fractions. The high-mol wt fraction contained both peptide and carbohydrate, and its activity was partially inhibited by proteolytic treatment. The low-mol wt fraction contained carbohydrate and could be replaced by a number of mono- and di-saccharides. Glucosamine and N-acetyl glucosamine were the most effective, whereas neutral sugars such as sucrose were less effective. Sucrose competed with glucosamine, so that lower levels of OSCN- were obtained with increasing amounts of sucrose. The sugars stimulated production of H2O2 by the saliva sediment. Production of H2O2 was greater in the presence of glucosamine than of neutral sugars. Also, the ratio of OSCN- accumulation to H2O2 production was greater in the presence of glucosamine. The results suggest that peroxidase-mediated antimicrobial activity is modulated by the carbohydrate composition of whole saliva and by certain protein and glycoprotein components.

Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes.

Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde-fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I-rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation.

The role of commercial bovine serum albumin preparations in the culture of one-cell rabbit embryos to blastocysts.

Normal bovine serum albumin (BSA) in a complete medium without energy substrates promoted growth of 1-cell embryos to hatched blastocysts. Defatted charcoal-treated BSA did not promote growth to the blastocyst stage but the addition of pyruvate or palmitic and oleic acids allowed blastocyst growth but not blastocyst hatching. Sodium dodecyl sulphate-gel electrophoresis showed that both the normal and defatted BSA samples were heavily contaminated by proteins other than albumin. Fractionation of the normal BSA on Sephadex G-200 indicated that the property of promoting complete blastocyst hatching was not due to the albumin but was associated with the higher mol. wt fraction of the BSA. Extraction of normal BSA with chloroform appeared to destroy the hatching-promoting ability as neither the residue after extraction nor defatted BSA to which the organic extractate had been added promoted complete blastocyst hatching. It is concluded that commercial BSA may have at least two effects on blastocyst growth: (1) energy provision via albumin-bound fatty acids, and (2) promotion of blastocyst hatching by a non-albumin component.

Increased Incidence of Tumor Takes to B16 Melanoma in C57BL/6J Mice Injected with B16 Products

The injection of either viable B16 melanoma cells, killed B16 cells, or B16 cell products increased the incidence of melanomas in C57Bl/6J mice inoculated with a threshold dose of B16 cells. In all cases the effect was seen whether the facilitating injection was at a site distant from the challenge inoculum or at the same site. Facilitation was seen with B16 cells and products both from in vivo tumors and from tissue culture. However, cells detached from tissue culture flasks with trypsin no longer had facilitating activity. Facilitating activity was found in concentrated cultured supernatants centrifuged at 100,000 X g for 1.5 hr and dialyzed. No activity was detected in the less than 10,000 mol wt fraction. Facilitation was not associated with a change in the time of tumor appearance nor with enhanced growth of B16 cells in culture.