Human teratoma cells share antigens with mouse teratoma cells.

Cytochalasin B inhibits the penetration of sperm nuclei into Urechis eggs without inhibiting sperm-induced egg activation. The acrosome reaction appears normal, and plasma membranes of the acrosomal tubule and egg become closely apposed. It is uncertain whether or not the drug blocks fusion of these membranes; however, sperm penetration cone formation is inhibited.     

Studies on an anophthalmic strain of mice. VI. Lens and cup interaction

In the embryology of the eye region in the anophthalmic strain of mice ($\text{ZRDCT}\text{Ch}$), development proceeds normally until Day 10 (26 somites). At this time a lens is induced, but it is smaller in size and may be improperly centered in the optic cup. Where the lens is centered in relation to the optic cup determines whether microphthalmia or anophthalmia will occur. Also, we observed that optic cup formation is different in normal control strains.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Reversal of insulin effects in fat cells may require energy for deactivation of glucose transport, but not deactivation of phosphodiesterase

The reversal of insulin effects on sugar transport and phosphodiesterase in fat cells was studied after arresting further actions of insulin with KCN, NaN₃, 2,4-dinitrophenol, or dicumarol. These agents rapidly lower the ATP concentration and concomitantly block the actions of insulin added later. Contrary to our expectation, the above inhibitors failed to initiate deactivation of the hormone-stimulated transport system. Instead, in the presence of the agents the transport system remained activated even after cells had been washed with an insulin-free buffer. This effect of the inhibitors was reversed when cells were washed with an inhibitor-free buffer containing glucose or pyruvate. The above inhibitors also blocked the deactivation of sugar transport stimulated by mechanical agitation. The effects of the inhibitors could not be explained by their possible effects on the basal transport activity, the intracellular urea space, or the cell count. The insulin-stimulated phosphodiesterase activity was rapidly lowered when cells were exposed to the above inhibitors. Apparently, these agents did not denature phosphodiesterase itself since the latter could be reactivated by insulin when inhibitor-treated cells were washed with a glucose-containing buffer. None of the above agents, except dicumarol, significantly inhibited phosphodiesterase activity in a cell-free system. It is suggested that the effects of insulin on sugar transport and phosphodiesterase are reversed by different mechanisms. ATP or metabolic energy may be involved in the deactivation of sugar transport, but not in that of phosphodiesterase.     

Resistance and susceptibility to infection in inbred murine strains. I. Variations in the response to thymic hormones in mice infected with Candida albicans

Nine inbred murine strains were either highly resistant or highly susceptible to intravenous challenge with 4 X 10(4) to 1 X 10(5) cells of Candida albicans. The resistant strains had the capacity to develop delayed footpad reactions on appropriate sensitization and challenge; the susceptible strains did not have this innate capacity. Administration of thymosin fraction 5 beginning on the day of infection greatly increased the resistance of the susceptible strains to infection, but decreased the resistance of the resistant strains. In contrast, thymosin fraction 5 enhanced the delayed footpad responses of resistant-sensitized mice to specific antigen, but did not have a detectable effect on the delayed footpad reactions of the susceptible strains. Reinfection of the two types of strains had different effects, in that, depending on the strain, resistance could be increased, decreased, or not influenced at all.     

Strain variation in interferon gamma production of BCG-sensitized mice challenged with PPD II. Importance of one major autosomal locus and additional sexual influences

Interferon-gamma (IFN-gamma) production in BCG-sensitized mice challenged with PPD was examined in the sera from BALB/c and C57BL/6 mice. C57BL/6 mice produce about ten times more IFN-gamma than BALB/c mice. Studies on F1, F2, and backcross generations indicate that one partially dominant autosomal locus is involved. Furthermore, females consistently produce more IFN-gamma than males in all of these crosses, though the X chromosome cannot be held responsible for this.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Synthesis and turnover of lipids in monolayer cultures of BHK-21 cells.

An aryl azide, 2,4-dinitro-5-fluorophenylazide, has been prepared and characterized with respect to the rate of nucleophilic displacement of the active fluorine by amine groups. This reaction occurs readily under mildly alkaline conditions and at temperatures below 30 °C, which makes it suitable for modifying free amine groups, even those of active enzymes. The resulting 2,4-dinitro-5-azidophenylamine derivative is a photoaffinity label. Absorption of light of wavelengths shorter than 450 nm causes generation of the highly reactive aryl nitrene that covalently inserts into its immediate environment.     

Phosphorylation associated with succinate decarboxylation to propionate in Ascaris mitochondria

Mono-, bis-, and tris-β-d-galactopyranosides of (2-(5-hydrazinocarbonylpentanamido)-2-(hydroxymethyl)-1, 3-propanediol were synthesized. Treatment of the glycosides with nitrous acid gave the corresponding acyl azides which were coupled to bovine serum albumin to form neoglycoproteins. These derivatives were tested for their inhibitory action on (i) the d-galactose binding by rabbit liver membrane, (ii) the corresponding binding by the isolated binding protein, and (iii) the corresponding uptake by intact rat hepatocytes. In these systems, the neoglycoprotein with attached tris-galacto was a better inhibitor than the bis derivative, which in turn was more effective than the mono derivative per each ligand. However, the order is reversed when the inhibitory action is expressed on the basis of d-galactosyl residue.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH₂ groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K⁺-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH₂ groups was studied after modification with the probes under various conditions and relating the inhibition of the K⁺-stimulated ATPase to the ATPase-dependent H⁺ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K⁺-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K⁺. Both the K⁺-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH₂ groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH₂ groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH₂ appeared to be associated with the K⁺-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH₂ groups of the K⁺-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K⁺-stimulated ATPase and dye uptake.     

Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Binding of naphthalene dyes to the N and A conformers of bovine α-lactalbumin

Contrary to earlier findings, monomeric native α-lactalbumin does bind naphthalene dyes such as ANS and TNS with marked enhancement of their fluorescence. Nanosecond decay measurements indicate there to be two dye binding sites per protein molecule with lifetimes of ca. 2 and 15 ns for ANS and 5 and 11 ns for TNS. The fluorescence titrations curves of α-lactalbumin with ANS and TNS reflect this site multiplicity, i.e., it was not possible to analyze such curves with a single K_diss. The apparent dissociation constants for binding of ANS and TNS to native bovine α-lactalbumin, as determined by an ultracentrifugal technique, ca. 950 and 900 μm, respectively, indicate that such binding is considerably weaker than previously supposed. The A conformer (metal ion-free form) of α-lactalbumin binds ANS and TNS more tightly than the N (native) form of the protein with marked fluorescence enhancement. The A conformer has two dye binding sites with lifetimes for ANS and TNS comparable with those seen with native protein.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

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.     

Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.